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Human Fungal Pathogens May 11-17

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Human Fungal Pathogens May 11-17 Powered By Docstoc
					Second FEBS Advanced Lecture Course
   Human Fungal Pathogens
  Molecular Mechanisms of Host-Pathogen
         Interactions and Virulence
          May 11-17, 2007
       La Colle sur Loup, France



          Evolution of pathogenic fungi
    Environmental sensing and morphogenesis
         Targets and antifungal strategies
             Host-fungus interactions



                     Organizers
     Christophe d’Enfert, Anita Sil & Steffen Rupp




                           1
                Organizing Committee
              Christophe d'Enfert (France)
                     Course Chair

 Anita Sil (USA)                    Steffen Rupp (Germany)
Course Vice-Chair                      Course Vice-Chair

                    Christine Dugast
                    Course Secretary




       International Scientific Advisory Board
                    Judith Berman (USA)
              Alistair Brown (United Kingdom)
                  Geraldine Butler (Ireland)
                   Melanie Cushion (USA)
                    Cameron Douglas (USA)
                  Paul Dyer (United Kingdom)
                       Concha Gil (Spain)
                  Gustavo Goldman (Brazil)
               Ken Haynes (United Kingdom)
                   Joseph Heitman (USA)
                 Bernhard Hube (Germany)
                  Alexander Johnson (USA)
                    Karl Kuchler (Austria)
                   Per Lungdahl (Sweden)
                    Aaron Mitchell (USA)
                    Luigina Romani (Italy)
              Dominique Sanglard (Switzerland)
                    Yue Wang (Singapore)




                           2
                   Sponsors

The Federation of European Biochemical Societies




                       3
       Important informations                                                    p. 4

       Indicative time table                                                     p. 9

       Program                                                                   p. 10

       Lecture abstracts                                                         p. 18

       Posters and workshop talks abstracts                                      p. 56

       List of participants                                                      p. 207

       Index of authors                                                          p. 228



                              IMPORTANT INFORMATIONS

Conference Secretariat
The secretariat takes care of all the administrative aspects of the conference : registration and
day-to-day organisation. The secretariat will be open at indicated hours on May 21 and May 22
and from 8.15 am to 8.45 am each morning before the meeting starts.


Note on reimbursements

Invited lecturers are expected to provide the organization with originals of their travel tickets
after the conference. Please check that the price - as well as your name - features on any ticket.
Please note that we can only reimburse your actual travel costs in the limit we have indicated
previously. E-tickets will be accepted.


Conference Facilities

Meeting room and equipment
The meeting room (Galoubet) is located near the main building of the centre. It is equipped with
a LCD projector, an overhead projector, and microphones. Two laptop computers will be
available (one PC, one Mac). Speakers in the plenary sessions, workshops and hot topic sessions
should plan to be in the meeting room 30 min. prior to their session to upload their presentation
onto these computers. It is therefore recommended that speakers bring their PowerPoint
presentation onto a CD-ROM or memory stick. Alternatively, speakers using a presentation
software other than powerpoint can bring their own laptop. Please note that equipment for 35
mm slides will not be provided at this meeting.
                                                4
Posters
Poster boards are located in room Miro, Kandinsky and Monnet. Posters should be posted on
Saturday May 12 and removed Wednesday May 16 before the session starts.
Participants should be at their poster on the following day:
Saturday May 12: Posters with the letter A
Sunday May 13: Posters with the letter B
Tuesday May 15: Posters with the letter C
Paper clips are provided to fix the posters on the boards.


Accommodation

General
Rooms have been booked for the nights of May 11 – May 16 inclusive (6 nights), with departure
after breakfast on Saturday May 17. All participants will stay in the VVF residence in single
accommodations (studio) or small apartments (two separate bedrooms and a shared bathroom).
Extra Nights
If you require extra accommodation in addition to the nights included in the conference booking,
you will need to contact VVF directly. Extra nights will be at your own expense. The price per
night, on a bed & breakfast basis, is EUR 27 in a twin/double room and EUR 47 in a single. An
extra meal costs about EUR 22.

Meals
Breakfast will be served buffet style from 7.30 am. Times for lunch and dinner are as shown in
the conference programme. Wine, mineral water and coffee are served at each meal. Additional
beverages are at participants’ own expense..


Site Services

Phonecalls
There are no telephones in the bedrooms. There are several public phones on the conference site.
These operate with calling cards that can be bought at the reception desk. A public phone
operating with coins is available in the main buidling.

Photocopies and Faxes
Photocopies may be made, and faxes sent and paid for via the VVF reception.

Computer Facilities and Internet Connexion
A WiFi internet access is available in the lobby..
There is an internet café in La Colle sur Loup.




                                                  5
Bank Facilities
The nearest bank, the Caisse d’Epargne is in La Colle sur Loup (2 km, 15 min. walking distance).
This bank provides an automat where withdrawals with an international credit card are possible.
Exchange of currencies is not possible at this bank.
Exchange of foreign currencies or withdrawal of Euros with an international credit card are also
possible at the Nice airport.

Means of Payment to the Conference Site
Credit cards are accepted by the VVF reception desk (not at the bar) and all currency payments
should be in Euro. It is not possible to change foreign currency at the centre.
Payment at the bar can only be made using an electronic bracelet. Participants will be proposed
this bracelet at the reception. The system must be loaded with a minimum credit of 10 Euros. The
credit that has not been used at the end of the conference will be refunded at check-out.


Leisure Activities and Tourism

Weather
Weather in the region of La Colle sur Loup in May is uncertain. Weather is normally sunny with
temperatures ranging from 18 to 22 °C during the day and 10 to 15°C during the night. However,
showers are also possible.

At the Conference Site
The venue is set within a 25 acres private pine-tree forest and provides numerous recreational and
leisure facilities: bar with terrace, TV room, American pool table, outdoor swimming pool,
volleyball and basketball courts, bowls pitch and 3 tennis courts.

In the Surrounding Area
The Nice back-country provides opportunities for hicking, rock-climbing and canyoning. La
Colle-sur-loup is close to the traditional village of Saint-Paul-de-Vence with the Maeght
Fundation displaying an impressive collection of modern art. It is also close to Nice with the
Matisse museum and the Chagall museum, to Vence with the Chapel of the Rosary imagined and
achieved by Henri Matisse, and to Vallauris with the Picasso museum. It is also close to Biot
famous for its glass workshop and Grasse famous for its perfume industry. Excursions will be
organized during the free afternoon of May 25.

Social Programme
A welcome drink will take place on Saturday May 21 and a special conference dinner and aperitif
will be served on the evening of Friday May 27.

Excursions will be proposed during the free afternoon of May 25. Three buses should be
organized going respectively to Saint-Paul-de-Vence and the Maeght Fundation, Nice, Biot and
Antibes. Particpants will be asked to select their preferred excursion at the meeting since there
will be limited availability for each excursion (53 persons each).



                                                6
Some Useful Information

Insurance
The meeting organizers do not provide insurance and do not take any responsibility for accidents
or illnesses that might occur during the week or in the course of travel to or from the meeting
place. It is therefore the responsibility of participants to check their health insurance
requirements.

Shopping Hours
The nearest shops (newspapers, toilet articles, etc ...) are at la Colle sur Loup, about 15 minutes
walk from the VVF. Major shopping can be done in Nice.

Opening times of shops in France in general are : Monday through Saturday : 09.00 to 12.00 and
14.00 to 19.00 but many close on Monday.

Extra Expenses
Please note that participants should pay the conference site for any additional nights outside the
nights covered by the registration fee.

All other additional expenses, e.g. drinks (other than those provided at meals), telephone calls,
tours, etc ... are also at participants’ own expense and should be paid upon check-out from the
VVF.




                                                7
8
                                                             INDICATIVE TIME-TABLE

            Friday May 11,        Saturday May 12,        Sunday May 13,       Monday May 14,        Tuesday May 15,         Wednesday May         Thursday May 17,
                 2007                   2007                   2007                2007                   2007                 16, 2007                  2007
7.00 am
                                       Breakfast              Breakfast             Breakfast             Breakfast               Breakfast            Breakfast

9.00 am
                                                              Session 2:
                                                                                                                               Session 5: Host-
                                  Session 1: Evolution     Pathogenic fungi,                            Session 4: Host-
                                                                                Session 3: Targets                                  fungus
                                  of pathogenic fungi -     environmental                            fungus interactions:
                                                                                  and antifungal                             interactions:… and
                                    genomics, sex and        sensing and                                  the pathogen                                 Departure
                                                                               strategies (20 min.                            the host responds
                                    poulation (20 min.    morphogenesis (20                            attacks... (20 min.
                                                                               intro + 5* 30 min.)                           (20 min. intro + 5*
                                   intro + 5* 30 min.)    min. intro + 5* 30                          intro + 5* 30 min.)
                                                                                                                                   30 min.)
                                                                 min.)

12.15 pm
                                         Lunch                  Lunch                Lunch                  Lunch                  Lunch

1.30 pm




                                                                                 Free afternoon




3.30 pm
                                     Coffee and Tea         Coffee and Tea                             Coffee and Tea          Coffee and Tea

4.00 pm


              Registration
                                    Selected talks 1       Selected talks 2                            Selected talks 3        Selected talks 4
                                     (7 * 20 min.)          (7 * 20 min.)                               (7 * 20 min.)           (7 * 20 min.)




7.00 pm
                 Dinner                  Dinner                Dinner                Dinner                 Dinner

8.30 pm


            Hot topic session                                                  Hot topics: systems
           Fungal Infections in      Poster session         Poster session     biology and fungal      Poster session        Dinner and Farewell
              the real life                                                         pathogens                                       Party


10.00 pm


            Wellcome Drinks




                                                                                9
                            PROGRAM

                           Friday May 11, 2007
3.00 pm     Registration

7.00 pm     Dinner

8.30 pm     Keynote lectures : Fungal infections in the real life

  8.30 pm   Christophe d’Enfert, Opening of the meeting

  8.40 pm   Markus Ruhnke, Charite University Medicine, Germany
            Current aspects on epidemiology and treatment of invasive fungal
            infections

  9.20 pm   Arturo Casadevall, Albert Einstein College of Medicine USA
            Origin of virulence in human pathogenic fungi

10.00 pm    Welcome drinks




                        Saturday May 12, 2007
7.30 am     Breakfast

8.00 am     Registration

9.00 am     Session 1: Evolution of pathogenic fungi - genomics, sex and population
            Chair: Joe Heitman

  9.00 am   Joe Heitman, Duke University, USA
            Introduction

  9.20 am   Melanie Cushion, University of Cincinatti College of Medicine, USA
            The transcriptome and genome of Pneumocystis carinii- strategies of
            a compatible parasite

  9.50 am   Geraldine Butler, University College Dublin, Ireland
            Evolution of mating in Candida species

  10.20 am Coffee break

  10.40 am Christina Hull, University of Wisconsin, USA
           The Sex: How Cryptococcus neoformans controls itself


                                     10
  11.10 am Jim Anderson, University of Toronto, Canada
           Incipient speciation by divergent adaptation and antagonistic
           epistasis in yeast

  11.40 am William Nierman, The Institute for Genomic Research, USA
           Comparative genomic analysis of sequenced Aspergilli

12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 1: Evolution of pathogenic fungi - genomics, sex and
            population
            Chairs: Geraldine Butler and Jim Anderson

  4.00 pm   P1 Ellisavet Nikolaou, University of Aberdeen, United Kingdom
            Phylogenetic Diversity of Fungal Osmotic and Cell Wall Stress-
            Signalling Proteins

  4.20 pm   P3 Laura Elena Carreto-Binaghi, Universidad Nacional Autónoma de
            México, Mexico
            Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats
            in Latin America

  4.40 pm   P4 Ute Zeidler, University of Salzburg, Austria
            Candida albicans orthologues of yeast meiotic genes have diverse
            functions in Candida biology

  5.00 pm   P7 Isabel Miranda, University of Aveiro, Portugal
            A unique CUG codon distribution in the Candida albicans genome

  5.20 pm   P10 Jennifer Reedy, Duke University, USA
            Creation of a Candida lusitaniae congenic strain pair and
            characterization of the mating type loci of Candida lusitaniae and
            Candida guilliermondii

  5.40 pm   P12 Ntombizamatshali Mtshali, University of Kwa-Zulu Natal, South
            Africa
            Characterization of fungal cell wall redox enzymes in the lichen
            Collema

  6.00 pm   P13 Toni Gabaldon, University of Valencia, Spain
            Using comparative genomics to predict function: recent applications
            and perspectives for elucidating pathways involved in pathogenesis.

7.00 pm     Dinner

8.30 pm     Poster session



                                    11
                        Sunday May 13, 2007
7.30 am     Breakfast

9.00 am     Session 2: Environmental sensing and morphogenesis
            Chair: Alistair Brown

  9.00 am   Alistair Brown, Aberdeen University, United Kingdom
            Introduction

  9.20 am   Bruce Klein, University of Wisconsin, USA
            The regulation of morphogenesis and virulence in dimorphic fungi

  9.50 am   Anita Sil, University of California San Francisco, USA
            Regulation of cell shape by temperature in Histoplasma capsulatum

  10.20 am Coffee break

  10.40 am Gustavo Goldman, Universidade de Sao Paulo, Brazil
           Transcriptome analysis of different factors that influence the
           Paracoccidioides brasiliensis Mycelium-to-Yeast transition

  11.10 am Christophe d’Enfert, Institut Pasteur, France
           Protein kinases and the regulation of morphogenesis in Candida
           albicans

  11.40 am Malcolm Whiteway, NRC Biotechnology Research Institute, Canada
           Rewiring of fungal transcriptional regulatory circuits


12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 2: Environmental sensing and morphogenesis
            Chairs: Per Ljungdahl and Gustavo Goldman

  4.00 pm   P19 Omar Loss, Imperial College London, United Kingdom
            Characterisation of calcium signalling by gene knock-out and
            microarray analysis in Aspergillus fumigatus

  4.20 pm   P31 Ana Garcera, Univesity of Lleida, Spain
            Glutathione transferases in Candida albicans and response against
            external stresses

  4.40 pm   P35 Francesco Citiulo, Trinty College, Ireland
            Purification and germination of chlamydospores produced by Candida
            albicans and Candida dubliniensis.



                                     12
  5.00 pm    P40 Andreas Roetzer, University of Vienna, Austria
             Living on a hostile ground: key transcription factors mediate stress
             response of Candida glabrata.

  5.20 pm    P47 Rachel Lane, University of Sheffield, United Kingdom
             Sec2p, the GEF for the Rab GTPase Sec4p localizes to the
             Spitzenkörper in the human fungal pathogen C. albicans.

  5.40 pm    P56 Siobhan Mulhern, University College Dublin, Ireland
             Analysis of the hypoxic response in Candida albicans

  6.00 pm    P48 Oliver Reuss, Würzburg University, Germany
             Environmental control of white-opaque switching in Candida albicans

7.00 pm      Dinner

8.30 pm      Poster session




                              Monday May 14, 2007
7.30 am      Breakfast

9.00 am      Session 4: Targets and antifungal strategies
             Chair: Dominique Sanglard

  9.00 am    Dominique Sanglard, Centre Hospitalo Universitaire Vaudois, Switzerland
             Introduction

  9.20 am    Theodore C. White, Seattle Biomedical Research Institute, USA
             The master sterol transcription factor UPC2 of Candida albicans
             regulates sterol genes and is regulated by sterols

  9.50 am    Carol Munro, University of Aberdeen, United Kingdom
             The fungal cell wall as a target for antifungals

  10.20 am Coffee break

  10.40 am Judith Berman, University of Minnesota, USA
           Contributions of aneuploidy and recombination to fluconazole
           resistance in Candida albicans

  11.10 am Steffen Rupp, Fraunhofer IGB, Germany
           Screening sytems to identify virulence mechanisms and new
           antifungals

  11.40 am Antonio Cassone, Istituto Superiore di Sanita, Italy
           Antifungal vaccines


                                      13
12.15 pm    Lunch

1.30 pm     Buses to Saint Paul de Vence and the Maegth Fundation, to Nice or to Biot
            and Antibes

7.00 pm     Dinner

8.30 pm     Hot topics: Systems Biology and Fungal Pathogens
            Chairs: Ken Haynes and Karl Kuchler
            Invited speakers: Guri Giaever and Jacky Snoep
            Chairs and speakers in this session will provide short overviews about how
            systems biology can make its way in the field of fungal pathogens. Participants
            are invited to actively participate in this discussion.

            Karl Kuchler, University of Vienna, Austria
            Introduction to the topic

            Guri Giaever, University of Toronto, Canada
            Understanding gene function and drug action

            Ken Haynes, Imperial College London, United Kingdom
            Network analysis of pH adaptation in Saccharomyces cerevisiae

            Jacky Snoep, University of Stellenbosch, South Africa
            Systems Biology of human fungal pathogens; potential and
            challenges for modeling




                        Tuesday May 15, 2007
7.30 am     Breakfast

9.00 am     Session 4: Host-fungus interactions: The pathogen attacks…
            Chair: Mike Lorenz

  9.00 am   Mike Lorenz, The University of Texas Health Science Center, USA
            Introduction

  9.20 am   Daniel Poulain, INSERM U799, France
            Expression of beta-1,2 mannoses at Candida albicans cell wall
            surface and host damage

  9.50 am   Guilhem Janbon, Institut Pasteur, France
            The genetics of the Cryptococcus neoformans capsule

  10.20 am Coffee break



                                      14
  10.40 am Nancy Keller, University of Wisconsin, USA
           Small molecules mediation of Aspergillus fumigatus virulence
           attributes

  11.10 am Concha Gil, Complutense University, Spain
           Integrated proteomic and genomic strategies bring new insight into
           Candida albicans response upon macrophage interaction

  11.40 am Brendan Cormack, John Hopkins University School of Medicine, USA
           NAD+ and the regulation of the EPA family of adhesins in Candida
           glabrata

12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 3: Targets and antifungal strategies
            Chairs: Judith Berman and Carol Munro

  4.00 pm   P88 Arnaud Firon, Institut Pasteur, France
            Conditional inactivation of SUN genes in Candida albicans reveals an
            essential and conserved cell-wall related function required at a late step
            of cell separation

  4.20 pm   P78 Moira Cockell, Centre Hospitalier Universitaire Vaudois, Switzerland
            Functional analysis of Pneumocystis carinii genes in
            Schizosaccharomyces pombe

  4.40 pm   P69 Alix Coste, Centre Hospitalier Universitaire Vaudois, Switzerland
            Mutations in drug resistance genes coupled with chromosome 5
            rearrangements mediate antifungal resistance in C. albicans

  5.00 pm   P77 Peter Staib, Centre Hospitalier Universitaire Vaudois, Switzerland
            Tetracycline-inducible expression of secreted aspartyl proteases in
            Candida albicans allows isoenzyme-specific inhibitor screening

  5.20 pm   P83 Oliver Bader, Georg-August University Göttingen, Germany
            Systematic investigation of cell wall modulations in clinical isolates of
            Candida glabrata

  5.40 pm   P87 Raquel Martinez-Lopez, Complutense University, Spain
            Immunoproteomic analysis of the protective response obtained from
            vaccination with C. albicans ecm33 mutant in mice

  6.00 pm   P82 Petra Schlick, Intercell AG, Austria
            Antigenome technology, a novel approach for the development of
            fungal subunit vaccines


7.00 pm     Dinner


                                     15
                    Wednesday May 16, 2007

7.30 am     Breakfast

9.00 am     Session 5: Host-fungus interactions: … and the host responds
            Chair: Neil Gow

  9.00 am   Neil Gow, Aberdeen University, United Kingdom
            Introduction

  9.20 am   Annika Scheynius, Karolinska Institute, Sweden
            Malassezia sympodialis – host-microbe interactions

  9.50 am   Alexander Johnson, University of California, USA
            Identification of Drosophila Gene Products Required for Phagocytosis
            of Candida albicans

  10.20 am Coffee break

  10.40 am Gordon Brown, University of Cape Town, South Africa
           Role of Dectin-1 in anti-fungal immunity

  11.10 am Anna Vecchiarelli, University of Perugia, Italy
           Purified capsular material of C. neoformans determines
           immunosuppression by direct engagement to Fc gamma RIIB
           receptor

  11.40 am Stuart Levitz, University of Massachussets, USA
           Immunity to Cryptococcus neoformans: O-Man, that’s sweet

12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 4: Host-fungus interactions
            Chairs: Nancy Keller and Anna Vecchiarelli

  4.00 pm   P101 Estelle Mogensen, Institut Pasteur, Paris
            Identification of genes involved in biofilm formation in Candida
            glabrata

  4.20 pm   P106 Franziska Lessing, Hans Knoell Institute, Germany
            Defence of Aspergillus fumigatus against reactive oxygen species
            mediated by Afyap1

  4.40 pm   P143 Constantin Urban, Max Planck Institute for Infection Biology,
            Germany
            Neutrophil extracellular traps capture and kill Candida albicans yeast
            and hyphal forms with a defined set of antimicrobial proteins.


                                    16
  5.00 pm      P118 Martin Zavrel, Fraunhofer IGB, Germany
               AUF gene-cluster potentially involved in host-pathogen interaction

  5.20 pm      P126 Steven Giles, University of Wisconsin, USA
               Sexual development in Cryptococcus neoformans is modulated by the
               steady-state concentration of endogenous reactive oxygen species

  5.40 pm      P128 Dervla Isaac, University of California, USA
               Identifying virulence factors in the fungal pathogen Histoplasma
               capsulatum.

  6.00 pm      P139 Attila Gacser, Albert Einstein College of Medicine, USA
               The PD1/PDL costimulatory pathway has a key regulatory role in
               Histoplasma capsulatum pathogenesis

6.20 pm        Anita Sil, concluding remarks

7.00 pm        Dinner and farewell party




                         Thursday May 17, 2007
From 7.30 am          Breakfast and departure




                                           17
ABSTRACTS

LECTURES




    18
       KEY-NOTE LECTURES

FUNGAL INFECTIONS IN THE REAL LIFE




                19
H11
Current aspects on epidemiology and treatment of invasive fungal infections
Markus Ruhnke
Medicine, Charité university medicine, Charitéplatz 1, Berlin 10117, Germany,
Phone: +4930450513036, FAX: +4930450513907, e-mail: markus.ruhnke@charite.de,
Web: www.charite.de

Substantial progress in the armamentarium of drugs for invasive fungal infections has been made.
Various new antifungals have demonstrated therapeutic potential. Currently, use of standard
antifungal therapy with amphotericin B desoxycholat (AmB-D) can be limited because of toxicity,
low efficacy rates, and eventually drug resistance. Liposomal formulations of amphotericin B have
a broad spectrum of activity against invasive fungi, such as Candida spp., C. neoformans, and
Aspergillus species. The liposomal AmB is associated with significantly less toxicity and good
rates of efficacy, which compare or exceed that of standard AmB-D. Whether these factors may
provide enough of an advantage to patients to overcome the increased costs of these formulations
is unclear (6). Moreover, adequate dosing of liposomal AmB is not finally evaluated. Three new
azole drugs have been developed, and may be of use in systemic fungal infections. Voriconazole,
ravuconazole, and posaconazole are triazoles, with broad-spectrum activity. Voriconazole has a
high bioavailability, and has been used with success in immunocompromised patients with
invasive fungal infections. In clinical phase II/III trials, efficacy has been shown in
immunocompromised patients suffering from oral and/or oesophageal candidosis as well as from
acute invasive or chronic invasive aspergillosis. In a randomized trial comparing AmB-D and
voriconazole for the treatment of acute invasive aspergillosis voriconazole proofed to be superior
to AmB-D and could be now regarded as the new standard for this disease. Ravuconazole has
shown efficacy in candidiasis in immunocompromised patients. Studies with posaconazole
indicated potential use in a variety of invasive fungal infections including oral / oesophageal
candidosis as well as rare infections such as mucormycosis. Echinocandins (Caspofungin,
Micafungin, Anidulafungin) are a new class of antifungals, which act as fungal cell wall beta-
(1,3)-D-glucan synthase enzyme complex inhibitors. Caspofungin is the first of the echinocandins
to receive approval for patients with invasive aspergillosis not responding or intolerant to other
antifungal therapies, and has been effective in patients with candidemia, oropharyngeal and
esophageal candidiasis.




                                               20
H12
Origin of virulence in human pathogenic fungi
Arturo Casadevall
Microbiology & Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue,
Bronx       NY     10461,      USA,      Phone: 718-430-3665,        FAX: 718-430-8711,       e-
mail: casadeva@aecom.yu.edu, Web: www.aecom.com
Arturo Casadevall. Albert Einstein College of Medicine, Bronx, NY Why are fungi pathogenic
for humans? The fact that human fungal infections are common, yet disease is rare implies that
humans have a natural resistance to fungal pathogens. Of the 1.5 million fungal species only a
very small minority have the capacity for mammalian virulence. This raises the fundamental
question of whether pathogenic fungal species are different, and if so, why. Human pathogenic
fungi are acquired from either other humans (e.g. Candida spp.) or the environment (e.g.
Cryptococcus neoformans, Histoplasma capsulatum, etc). Fungal diseases caused by fungi
acquired from other humans usually reflect a disruption of the host-microbe equilibrium. As an
example, C. albicans usually causes disease in the setting of antibiotic use, immunosuppression, or
compromise integument and mucosal membranes. In contrast, environmental fungi cause disease
in both normal or immunocompromised hosts in situations that can involve impaired immunity or
unusual exposures with large inocula. One example of environmentally acquired fungal pathogens
is C. neoformans, a soil microbe that causes disease in individuals with impaired immune systems
or after infection with large inocula. C. neoformans has been a model system for understanding
how virulence emerges and is maintained in the environment. One of the remarkable aspects of
this organism is that is has a unique intracellular strategy. Recent findings indicate that this
organism can escape from macrophages using a novel and unique exit strategy that has no
counterparts among known intracellular microbes. Comparison of the interactions of C.
neoformans with amoebae and macrophages reveals striking similarities suggesting that virulence
for mammals may be, at least in part, a result of selection pressures in soil from predatory
amoeboid organisms. This raises the hypothesis that virulence in soil pathogenic fungi arouse by
chance selection from biological and physical processes in the environment. The concept of
‘accidental virulence’ provides a new approach to understanding virulence factors and considering
fundamental questions of microbial virulence.




                                                21
          SESSION 1

EVOLUTION OF PATHOGENIC FUNGI
 GENOMICS, SEX AND POPULATION




             22
S11
The transcriptome and genome of Pneumocystis carinii- strategies of a compatible
parasite
Melanie     T. Cushion1,    A.    George Smulian2,     Bradley Slaven1,   Tom Sesterhenn1,
                3              4                5                  6
Jonathan Arnold , Chuck Staben , Alexsey Porollo , Rafal Adamczak and Jarek Meller7
1
  Internal Medicine, Division of Infectious Diseases, University of Cincinnati College of
Medicine, 231 Albert Sabin Way, Cincinnati OH 45140-0560, United States, Phone: +1 (513)
861 3100 ext 4417, FAX: +1 (513) 475 6415, e-mail: melanie.cushion@uc.edu,
Web: http://www.pathobiology.uc.edu/html/disea_immun_cushion.html 2 VAMC, 3200 Vine
St. Cincinnati, OH 45220 3 University of Georgia, Athens, GA 4 University of Kentucky,
Lexington, KY 5 University of Cincinnati, Dept. Environmental Health, Cincinnati, OH 6
Univeristy of Cincinnati,Dept. Env. Health, Cincinnati,OH 7 University of Cincinnati, Dept.
Env. Health, Cincinnati, OH

Members of the fungal genus Pneumocystis can cause a lethal pneumonia in mammals with
debilitated immune systems. Evidence suggests they survive in immunologically intact hosts and
are host-dependent fungi. Considered to be ascomycetes, Pneumocystis were placed in the
subphylum Taphrinomycotina, Order Pneumocystidales, Class Pneumocystidomycetes, Family
Pneumocystidaceae, Genus Pneumocystis. The Taphrinomycotina are a paraphyletic group of
organisms and the identity of the closest extant relative to the genus Pneumocystis is not clear.
Pneumocystis cannot be grown outside the mammalian lung, hindering almost every aspect of its
research. An EST and genome sequencing project were initiated to better understand their basic
biological and potential pathological processes. A 1085 member unigene set and the unassembled
reads were analyzed by BLAST to reveal 56% had identity to existing polypeptides at e<10-6 ,
with most(70%)to fungal or P. carinii sequences. KEGG Pathway mapping showed the homologs
represented most standard metabolic pathways and cellular processes, with carbohydrate
metabolism garnering the most transcripts (e.g.glycolysis, pentose phosphate pathway,TCA
cycle). An FBP1 gene homolog was not identified and no homologs in the glyoxylate pathway or
associated peroxisomal receptors were found, suggesting Pneumocystis may be unable to
synthesize glucose from non-carbohydrate precursors or utilize 2-carbons sources; the former may
be a potential fungal virulence factor. Several gene homologs specific for a sexual mode of
reproduction and meiosis were identified including pheromone receptors, signal transduction
components, chromatin silencing and cell cycle associated genes. Pneumocystis do not contain
ergosterol and are thought to salvage cholesterol from the host, but the presence of homologs in
sterol biosynthesis suggests this pathway may be operational, at least during some part of their life
cycle. Genes encoding the major surface glycoprotein family (MSG), heat shock, and proteases
were the most abundantly expressed of known P. carinii genes. MSG proteins have been
implicated in adhesion to host cells and may function as adhesins in the mating process. The
apparent presence of many metabolic pathways in P. carinii, sexual reproduction within the host,
and other known characteristics suggest members of the genus Pneumocystis are distinct from
other “opportunistic” fungal pathogens and are instead, adapted parasites and their relationship
with the host may be a compatible one.




                                                 23
S12
Evolution of mating in Candida species
Geraldine Butler and Mary Logue
Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland, Phone: +353-1-
7166885, FAX: +353-1-2837211, e-mail: geraldine.butler@ucd.ie,
Web: http://www.ucd.ie/sbbs/staff/butler_geraldine/index.html

We have carried out a comparative analysis of mating and meiotic pathways in related Candida
species, facilitated by the availability of the genome sequences of Candida tropicalis, C.
lusitaniae, C. guilliermondii and Lodderomyces elongisporus from the Broad Institute, C.
dubliniensis and C. parapsilosis from the Sanger Institute and C. albicansand Debaryomyces
hansenii in the public domain. The mating type loci (MTL) of C. albicans, C. dubliniensis and C.
tropicalis are very similar. The MTL of C. parapsilosis, C. guilliermondii, C. lusitaniae and the
fused locus of D. hansenii however are missing at least one component of the a1/alpha2 repressor.
L. elongisporus does not have any mating type locus. We identified the a-pheromone (MFA) from
a comparison between C. albicans and C. dubliniensis. The signal transduction pathway (from
pheromone to transcription factor) is generally well conserved in the species examined. However,
L. elongisporus is missing MFA, two proteins required for processing of MFA, and the a-
pheromone receptor (STE3). In addition, alpha-factor from C. parapsilosis is a poor inducer of
expression of genes. We cannot identify any MTLalpha idiomorph in C. parapsilosis, nor in an
initial survey of the related species C. orthopsilosis and C. metapsilosis. The C. albicans genome
contains orthologues of many genes required for meiosis in S. cerevisiae, apart from ZIP1and
RED1 (components of the synaptonemal complex). These are also missing from the genomes of
the other species. In addition, the sexual yeasts C. guilliermondii and C. lusitaniae are missing
other components required for the formation of the synaptonemal complex, Dmc1-dependent
meiotic recombination, and crossover interference. In conclusion, we suggest that the paradigm of
white/opaque switching and the cryptic sexual cycle described for C. albicans is also applicable to
C. dubliniensis, and possibly C. tropicalis. In the lineage leading to C. parapsilosis the mating
pathway has degenerated, and is almost completely missing in L. elongisporus. Whereas all the
species analysed may have the ability to undergo meiotic recombination, the mechanism involved
has diverged dramatically in the lineage leading to C. lusitaniae and C. guilliermondii.




                                                24
S13
Sex: How Cryptococcus neoformans controls itself
Brynne C. Stanton, Mark W. Staudt and Christina M. Hull
Dept. of Biomolecular Chemistry, University of Wisconsin, Madison, 1300 University Ave.
587 MSC, Madison WI 53706, United States, Phone: 608-265-5441, FAX: 608-262-5253, e-
mail: cmhull@wisc.edu

Cryptococcus neoformans is an opportunistic fungal pathogen that affects primarily
immunocompromised individuals. Infections with C. neoformans are thought to be caused by
spores, which can result from sexual development. During sexual development haploid a and
alpha cells fuse and initiate a process controlled by the homeodomain proteins Sxi1alpha and
Sxi2a. Our experiments are focused on determining the molecular mechanisms by which
Sxi1alpha and Sxi2a control sexual development and, ultimately, spore production. Our
hypothesis is that Sxi1alpha and Sxi2a control sexual development by directly regulating the
transcription of key targets to specify the dikaryotic state. To identify targets of Sxi1alpha and
Sxi2a, we are taking several integrated approaches, including using C. neoformans microarrays to
identify genes regulated by Sxi1alpha and Sxi2a. Targets of interest are being tested for direct
regulation by Sxi1alpha and/or Sxi2a using chromatin immunoprecipitations. Promoter sequences
from both bioinformatic and microarray targets are being tested using in vitro DNA binding
experiments with purified Sxi1alpha and Sxi2a proteins. Preliminary DNA binding studies show
that the homeodomain regions of Sxi1alpha and Sxi2a bind specifically to the promoter sequences
of many microarray targets, including a putative homolog of the clampless (CLP1) gene.
SXI1alpha and SXI2a are the first identified sexual cycle regulators in C. neoformans, and the
characterization of the pathway they control will reveal how sexual development and spore
formation occur in C. neoformans.




                                               25
S14
Incipient speciation by divergent adaptation and antagonistic epistasis in yeast
James B, Anderson
Cell and Systems Biology, University of Toronto, 3359 Mississauga Road North, Mississauga
ON L5L 1C6, Canada, Phone: 905 828-5362, FAX: 905-828-3792, e-
mail: janderso@utm.utoronto.ca

Establishing the conditions that promote the evolution of speciation has long been a goal in
evolutionary biology and is relevant to fungi causing disease in their hosts. The fundamental link
between divergent adaptation and reproductive isolation though genetic incompatibilities has been
predicted, but not directly demonstrated experimentally. I will first describe our experiments
following the evolution of resistance to the antifungal agent fluconazole in populations of Candida
albicans and Saccharomyces cerevisiae in which independently-evolved mechanisms of resistance
were antagonistic to one another, suggesting a possible route to full reproductive isolation - and
speciation. Do these conclusions apply to divergent adaptation in general? After replicate
populations of S. cerevisiae adapted over 500 generations to two divergent environments, high salt
or low glucose, we consistently observed the evolution of two forms of postzygotic isolation:
reduced rate of mitotic reproduction and reduced efficiency of meiotic reproduction. This
divergent selection resulted in greater reproductive isolation than parallel selection, as predicted
by ecological speciation theory. Our experimental system allowed the first controlled comparison
of the relative importance of ecological isolation through environment-specific adaptation and
genetic isolation through antagonistic epistasis. For mitotic reproduction, hybrid inferiority was
conditional upon the selective environments and was both ecological and genetic in basis. In
contrast, isolation associated with meiotic reproduction was unconditional and was caused solely
by genetic mechanisms. Overall, our results show that adaptation to divergent environments
promotes the evolution of reproductive isolation through antagonistic epistasis, providing
evidence of a plausible common avenue to speciation and adaptive radiation in nature. I will
discuss how these results may apply to fungi causing disease in their hosts.




                                                26
S15
Comparative Genomic Analysis of Sequenced Aspergilli
William Nierman1, Natalie Fedorova1, Vinita Joardar1, Jonathan Crabtree1, Rama Maiti1,
Paolo Amedeo1, Jennifer Wortman1, Elaine Bignel2 and Geoffrey Turner3
1
  , The Institute for Genomic Research, 9712 Medical Center Drive, Rockville MD 20850,
USA, Phone: 301-795-7559, FAX: 301-838-3009, e-mail: wnierman@tigr.org,
Web: www.tigr.org 2 Imperial College, London, UK 3 University of Sheffield, Sheffield, UK

Comparative genomic analyses of two strains of Aspergillus fumigatus, and single strains of A.
clavatus, and Neosartorya fischeri revealed an orthologous “core” shared by all three aspergillus
species, and species-specific components, which comprise from 9% to 15% of each genome. In
addition to information processing and primary metabolism, core genes have been linked to sex,
invasive aspergillosis, and allergic reactions. In contrast species-specific genes are often involved
in unknown functions, secondary metabolite biosynthesis and detoxification. They differ
significantly in their gene structure from core genes and tend to cluster in evolutionary labile
chromosomal regions similar in some ways to prokaryotic genomic islands. The origin of most
species-specific genes appears to involve duplication followed by rapid diversification or gene
loss, but no horizontal gene transfer. Specifically, evolution of secondary metabolism gene
clusters has been linked to de-novo assembly, segmental duplication, translocation, and
accelerated diversification. Comparison of the Af293 and CEA10 strains revealed up to 200 strain-
specific genes (~2%) and numerous sequence polymorphisms including genes presumably
associated with self/non-self recognition during hyphal fusion (heterokaryon incompatibility).




                                                 27
               SESSION 2

ENVIRONMENTAL SENSING AND MORPHOGENESIS




                  28
S21
The regulation of morphogenesis and virulence in dimorphic fungi
Bruce Klein
Pediatrics and Medical Microbiology, University of Wisconsin-Madison, 600 Highland
Avenue, Madison WI 53792, USA, Phone: 608-263-9217, FAX: 608-263-6210, e-
mail: bsklein@wisc.edu, Web: http://www.medmicro.wisc.edu/department/faculty/klein.html

The signature feature of systemic dimorphic fungi – a family of six primary fungal pathogens of
humans – is a temperature-induced phase transition. These fungi grow as a mold in soil at ambient
temperature and convert to yeast after infectious spores are inhaled into the lungs of a mammalian
host. Seminal work 20 years ago established that a temperature-induced phase transition from
mold to yeast is required for virulence. Several yeast-phase specific genes, identified one-by-one
and studied by reverse genetics, have revealed mechanisms by which the phase transition
promotes disease pathogenesis. Transcriptional profiling of microarrays built with genomic
elements of Histoplasma capsulatum and ESTs of Paracoccidioides brasiliensis that represent
partial genomes has identified 500 genes and 328 genes, respectively, that are differentially
expressed upon the phase transition. The genomes of most of the dimorphic fungi are now in
varying stages of being sequenced. The creation of additional microarrays and the application of
new reverse genetic tools promise fresh insight into the genes and mechanisms that regulate fungal
sensing of environmental stress, morphogenesis, and pathogenesis. The use of insertional
mutagenesis with Agrobacterium tumefaciens has uncovered a hybrid histidine kinase that
regulates dimorphism and pathogenicity in Blastomyces dermatitidis and H. capsulatum. Two-
component signaling appears to be a common strategy for model and pathogenic fungi to sense
and respond to environmental stresses.




                                               29
S22
Regulation of cell shape by temperature in Histoplasma capsulatum
Van Nguyen, Rachael Hanby, Dana Gebhart and Anita Sil
Microbiology and Immunology, University of California San Francisco, 513 Parnassus, S-469,
UC Medical Center, San Francisco CA 94143-0414, United States, Phone: 415 502-1805,
FAX: 415 476-8201, e-mail: sil@cgl.ucsf.edu, Web: http://www.ucsf.edu/micro/sil/index.htm

The long-term goal of our research is to determine how environmental signals regulate
morphology and virulence in the fungal pathogen Histoplasma capsulatum. H. capsulatum grows
in a filamentous mold (mycelial) form in the soil and a budding yeast form in the host. The
transition from the mycelial form to the yeast form, which occurs when cells are shifted to 37
degrees Celcius, is thought to be essential for virulence. Although temperature is known to be a
key signal that triggers the conversion between the two forms of H. capsulatum, little is known
about the molecular mechanism of how temperature regulates morphology. We are using a
combination of genetics and functional genomics to identify factors that regulate morphology. We
have identified three genes (named RYP1, RYP2, and RYP3 for Required for Yeast-Phase Growth)
that are necessary for cells to grow in the yeast form at 37 degrees Celcius. Mutation of any of
these genes results in constitutive mycelial growth independent of temperature. RYP1 encodes a
conserved fungal protein that appears to be a transcriptional regulator of the yeast-specific gene
expression program. Both Ryp1 transcript and protein accumulate preferentially at 37 degrees, and
microarray analysis shows that Ryp1 is necessary for the expression of most genes that are
normally expressed at this temperature. Similarly, Ryp2 and Ryp3 transcripts are differentially
expressed at 37 degrees, and their transcript accumulation is significantly reduced in the ryp1
mutant. Ryp2 and Ryp3 are homologous to the veA family of proteins from Aspergillus, which
has recently been shown to regulate gene expression. We are characterizing how these regulatory
proteins respond to temperature to stimulate yeast-phase growth.




                                               30
S23
Transcriptome analysis of different factors that influence the Paracoccidioides brasiliensis
Mycelium-to-Yeast transition
Gustavo Goldman
Ciencias Farmaceuticas, FCFRP, Universidade de Sao Paulo, Av. do Cafe S/N, Ribeirao Preto
SP 14040903, BRAZIL, Phone: +55(16)36024280, FAX: +55(16)36024280, e-
mail: ggoldman@usp.br, Web: goldman.fcfrp.usp.br

Paracoccidioides       brasiliensis   is    a    thermodimorphic       fungus      associated      with
paracoccidioidomycosis (PCM), a prevalent systemic mycosis in South America. In humans,
infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and
transform into the yeast parasitic form. Temperature and the inability to assimilate inorganic
sulphur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y)
dimorphic transition. M-Y transition can be inhibited by adding dibutyryl-cAMP, cyclosporine,
and beta-estradiol to the culture medium. Thus, the mycelium-to-yeast transition is of particular
interest because conversion to yeast is essential for infection. We have used a P. brasiliensis
biochip, carrying sequences of 4,692 genes from this fungus to monitor gene expression at several
time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). One gene, encoding
4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD) was highly overexpressed during the
mycelium-to-yeast differentiation and the use of NTBC (as well as NTBC derivatives), a specific
inhibitor of 4-HPPD activity was able to inhibit growth and differentiation of the pathogenic yeast
phase of the fungus in vitro. We also compared the M-Y transition in minimal medium with
complete medium. Our results showed that about 95 % of the genes in our microarray are mainly
responding to the temperature trigger, independently of the media where took place the M-Y
transition. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast
form, we decided to characterize the mRNA accumulation of several genes involved in different
aspects of both organic and inorganic sulphur assimilation. Our results suggest that although P.
brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition
and Y growth, the fungus can somehow use both organic and inorganic pathways during these
growth processes.

Financial support: FAPESP and CNPq, Brazil




                                                  31
S24
Protein kinases and the regulation of morphogenesis in Candida albicans
Christophe d'Enfert1, Sophie Goyard1, Claire Naulleau1, Hélène Munier-Lehman2,
Christine Laurent3, Ismaïl Iraqui4, Hyunsook Park5, Norma Solis5, Patrick Schwartz4,
Françoise Dromer4, Guilhem Janbon4 and Scott Filler5
1
  Unité Biologie et Pathogénicité Fongiques, Institut Pasteur, 25 rue du Docteur Roux, Paris
75015, France, Phone: +33 1 40 61 32 57, FAX: +33 1 45 68 89 38, e-
mail: denfert@pasteur.fr, Web: http://www.pasteur.fr/bpf 2 Unité de Chimie Organique,
Institut Pasteur, Paris, France 3 Plate-Forme Protéomique, Institut Pasteur, Paris, France 4 Unité
de Mycologie Moléculaire, Institut Pasteur, Paris, France 5 Harbor-UCLA Medical Center,
Torrance, USA

The ability to switch between different morphological forms – yeast, hypha and pseudo-hypha
- is central to the pathogenesis of Candida albicans. Different signaling cascades have been
implicated in the positive regulation of the yeast-to-hypha switch. This switch is also
negatively regulated by a Tup1-dependent process. Protein kinases play a central role in the
signaling cascades that regulate morphogenesis. This will be illustrated through two examples:
the Tpk1 and Tpk2 catalytic subunits of the cAMP-dependent protein kinase A (PKA) and the
Yak1 kinase. PKA is well known for relaying hypha-inducing cues towards the main
transcriptional regulator of morphogenesis, Efg1. Here, we will show how alleles of TPK1 and
TPK2 have been engineered so that their products can be specifically inhibited by an analog of
ATP, 1-NMPP1. Strains of C. albicans that express these mutant alleles can be used to
demonstrate that inhibition of Tpk1 and Tpk2 is synthetically lethal. Also, the impact of 1-
NMPP1 on the virulence of C. albicans strains expressing these inducible protein knockouts in
animal models of candidosis will be presented. The C. albicans Yak1 kinase was investigated
because of its role in biofilm formation in the pathogenic yeast, Candida glabrata. Inactivation
of C. albicans YAK1 results in defects in the yeast-to-hypha transition under both Efg1-
dependent and -independent hypha-inducing conditions. This result, along with additional data
obtained using a 1-NMPP1-sensitive isoform of Yak1, and serum-dependent induction of
hypha formation, indicates that Yak1 is involved in both the initiation and the maintenance of
hyphal growth in vitro. Surprisingly however, Yak1 is not required for virulence of C.
albicans, suggesting that hypha-inducing cues found in mice activate a Yak1-independent
signaling pathway. Yak1 does not appear to be linked directly to the cAMP-PKA signaling
pathway. Results of transcriptomic and phosphoproteomic approaches that are currently being
used to investigate the function of C. albicans Yak1 will be presented.




                                                 32
S25
Rewiring of fungal transcriptional regulatory circuits
Malcolm Whiteway, Herve Hogues, Hugo Lavoie, Mike Martchenko, Maria Mangos,
Andre Nantel and Adnane Sellam
Health Sector, NRC Biotechnology Research Institute, 6100 Royalmount Ave., Montreal QC
H4P 2R2, Canada, Phone: 001 514 496 6146, FAX: 001 514 496 6213, e-
mail: malcolm.whiteway@cnrc-nrc.gc.ca

An important component of cellular control is coordinate transcriptional regulation of genes
encoding functionally related activities. Intriguingly, different organisms can control similar
cellular processes by distinct transcriptional circuits. We have been comparing transcriptional
control in regulatory circuits of C. albicans and the model yeast S. cerevisiae. Although both C.
albicans and S. cerevisiae activate the Leloir pathway genes involved in galactose metabolism in
response to the presence of galactose, the regulatory circuits are distinct. In S. cerevisae this
process is under the positive control of the Gal4p transcription factor acting through a Gal4p
binding motif. In C. albicans the identical motif is found upstream of genes implicated in
glycolysis, and is bound by a transcription factor with strong sequence similarity to yeast Gal4p in
the DNA binding region. Instead of this Gal4-like circuit, the Leloir pathway genes of C. albicans
contain a distinct upstream palindrome that acts through the Cph1p transcription factor. Similar
rewiring events are found in the control of ribosomal protein gene expression, and peroxisomal
gene expression, suggesting the distinct ecological niches of these two yeasts have resulted in
dramatic changes in their regulatory wiring.




                                                33
            SESSION 3

TARGETS AND ANTIFUNGAL STRATEGIES




               34
S31
The master sterol transcription factor UPC2 of Candida albicans regulates sterol genes
and is regulated by sterols.
Theodore C. White, Jia Song, Brian Oliver, Peter Silver, Sam Hoot, Theresa Richards and
Chelsea Samaniego
Pathobiology, Seattle Biomedical Research Institute, 307 Westlake Ave N Ste. 500, Seattle
WA 98109-5219, United States, Phone: 1 206 256 7344, FAX: 1 206 256-7229, e-
mail: white@sbri.org

Erg11p is a cytochrome P450 enzyme involved in the biosynthesis of ergosterol, the major sterol
in fungal cells. Erg11p is also the target of azoles, which are a major type of antifungal drug in
clinical use against fungal infections. ERG11 is transcriptionally upregulated in the presence of
azoles and other drugs that affect the ergosterol biosynthetic pathway. Promoter deletion and
mutation has identified a specific region of the ERG11 promoter that is important for the ERG11
response to drugs. This region includes an inverted repeat that is recognized by the master sterol
regulator Upc2p. UPC2 encodes a Zn2Cys6 transcription factor with homology to the
Saccharomyces cerevisiae genes UPC2 and ECM22. These transcription factors may regulate
sterol metabolism in yeasts in a manner similar to sterol metabolism in mammalian cells by the
SREBP transcription factor. Deletion of the Candida albicans UPC2 gene results in cells that are
hypersusceptible to drugs effecting ergosterol biosynthesis as well as drugs that effect cell wall
structure and function. The deletion strain shows decreased sterol uptake and decreased sterols in
the plasma membrane. Northern blot and microarray analysis indicates that the UPC2 deletion
strain does not increase transcription of several ERG biosynthetic genes including ERG11 in the
presence of azole drugs. Promoter sequence analyses indicate that most if not all ERG genes, and
the UPC2 gene itself, may be regulated by UPC2. Current work is centered on the analysis of the
UPC2 gene promoter to identify transcription factors that activate this gene. In addition, the
domains of UPC2 are being investigated for their effect on sterol metabolism and gene expression.
Finally, localization and processing of the UPC2 gene and specific domains is being investigated
in the presence and absence of azole drugs. These results establish the UPC2 gene as an important
regulator of ergosterol metabolism, including biosynthesis and uptake, and they establish UPC2 as
an important and significant component of the fungal cell’s response to antifungal drugs.




                                               35
S32
The fungal cell wall as a target for antifungals
Carol Munro, Louise Walker and Neil Gow
School of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD,
United Kingdom, Phone: +44 (0) 1224555927, FAX: +44 (0) 1224555844, e-
mail: c.a.munro@abdn.ac.uk, Web: http://www.abdn.ac.uk/ims/staff/details.php?id=c.a.munro

The enzymes that synthesise chitin and 1,3-beta-D glucan, the essential structural components of
the fungal cell wall, are unique to fungi and are attractive targets for novel therapies. Chitin is
synthesised by a family of chitin synthase isoenzymes and although chitin synthase inhibitors exist
they are not potent against whole cells in vivo and so chitin synthesis remains an unexploited
potential target. Echinocandins that inhibit 1,3-beta-D glucan synthesis are a recent addition to the
antifungal armoury. Assembly of the fungal wall is a dynamic process and cell wall defects
activate compensatory mechanisms to maintain a robust cell wall. We have shown that 1,3-beta-D
glucan synthase inhibitors including caspofungin and other candins, activate C. albicans chitin
synthesis in an attempt to restore cell wall integrity. The role of the four C. albicans chitin
synthases Chs1, 2, 3 & 8 in the response to echinocandins has been examined. Promoter-lacZ
reporter fusions showed expression from the CHS1, CHS2 and CHS8 promoters was upregulated
when cells were treated with different echinocandins. There was no change in expression from the
CHS3 promoter. Treatment with caspofungin at sub-MIC levels also increased chitin synthase
activity and cell wall chitin content. The PKC, calcineurin-Crz1 and HOG signalling pathways
were involved in the response to caspofungin The increase in chitin content was dependent on
Chs3p and the PKC pathway, whereas the increase in chitin synthase activity was due to Chs2p,
Chs8p and the PKC, HOG and Calcineurin pathways. Western blot analysis with anti-phospho
p44/p42 specific antibodies confirmed that Mkc1, the MAP kinase of the PKC pathway, is rapidly
phosphorylated in response to caspofungin. Pretreating C. albicans with CaCl2 and Calcofluor
White (CFW) or with glucosamine, which elevates chitin content, led to reduced susceptibility to
caspofungin. But CFW and chitin synthase inhibitors acted synergistically with caspofungin to
inhibit growth. Even caspofungin resistant strains with point mutations in the FKS1 gene were
unable to survive this combination of cell wall inhibitors. Together these approaches have
provided a better understanding of the mechanisms controlling chitin synthesis and how these are
coordinated to enable cells to respond to treatments with antifungal drugs. Combination therapies
that target both chitin and glucan biosynthesis have significant potential in treating Candida
infections.




                                                 36
S33
Contributions of aneuploidy and recombination to fluconazole resistance in Candida
albicans
Judith Berman, Anna Selmecki and Anja Forche
Genetics, Cell Biology & Development, University of Minnesota, 321 Church St. SE, 6-160
Jackson H, MInneapolis MN 55455, USA, Phone: 612-625-1971, FAX: 612-626-6140, e-
mail: jberman@umn.edu,
Web: http://www.cbs.umn.edu/labs/berman/index.htmhttp://www.cbs.umn.edu/labs/berman/in
dex.htm
Genomic instability and aneuploidy are common in Candida albicans. Using comparative genome
hybridization (CGH) microarrays, we found a strong correlation between aneuploidy and azole
resistance1. We identified a segmental aneuploidy of chromosome 5L (Ch5L) DNA, due to the
formation of an isochromosome (a chromosome with two identical arms), in many fluconazole
resistant (FluR) strains. Southern hybridization to CHEF gels and SNP array analysis 1 shows that
Isochromosome 5L (i(5L)) formation is often accompanied by loss of heterozygosity (LOH) along
Ch5L. Microarray analysis of i(5L) strains show that most genes on Ch5L exhibit increased
expression and may contribute to FluR. Several well characterized genes involved in drug
transport, ERG11 and TAC1, are present within the isochromosome and thus are present in
increased gene copies. Deletion of one or two extra copies of these genes in an i(5L) strain
background has only partial effects on FluR, suggesting that increased copies of each gene alone is
not sufficenbt to account for the high level of resistance seen in the i(5L) strain. Telomere-
mediated chromosome truncations of Ch5L suggest that ERG11 and TAC1 together account for
much of the resistance seen in i(5L) strains. Importantly, i(5L) is relatively stable under rich
culture conditions, loss of i(5L) does occur and always correlates with a loss of FluR. The effects
of aneuploidy on gene copy number, gene expression level, LOH, and acquired azole resistance
will be discussed. 1. Selmecki et al. (2006) Science 313: 367-370. 2. Forche et al. 2005. Eukaryot
Cell 4:156-65.




                                                37
S34
Screening systems to identify virulence mechanisms and new antifungals
Steffen Rupp
Molecular Biotechnology, Fraunhofer IGB, Nobelstr. 12, Stuttgart 70569, Germany,
Phone: +49 (0) 711 970 4045, FAX: +49 (0) 711 970 4200, e-mail: rupp@igb.fhg.de,
Web: www.fraunhofer.igb.de

The sequencing of many different genomes opens promising opportunities for diagnostics and
target screening. Knowledge of the DNA- and protein sequences of an organism allows the
precise, fast and efficient identification of pathogens such as fungal species pathogenic to human,
as well as the identification of putative targets or resistance mechanisms. Gene expression profiles
have been very instrumental in unraveling mechanisms of Candida albicans involved in virulence
as well as elucidating resistance mechanisms. The detection of point mutations (SNPs) causing
resistance using array technologies can also be helpful to identify a preexisting resistance against a
given drug in a strain causing an infection. Diagnostic arrays to detect these SNPs are currently
developed. To study the response of C. albicans adhering to different surfaces on the
transcriptional level we have established an in vitro adhesion assay exploiting confluent
monolayers of the human cell lines or primary cells. C. albicans very efficiently adheres to these
epithelia growing as hyphae. Using whole-genome DNA microarrays we found that transcriptional
profiles of C. albicans adhering to different cellular surfaces, although very similar, still differ
significantly from those of Candida cells adhering to plastic surfaces. Differences are increasing
when comparing C. albicans cells either growing in an adherent manner or in suspension culture
pointing out potential novel targets. The putative targets identified through our transcriptome and
proteome analyses are used to develop high throughput assays for the screening of compound
libraries. Currently we screen combinatorial chemical libraries using assays that simultaneously
measure both antifungal activity as well as the toxicity of the substances in relation to human cells.




                                                 38
S35
Antifungal vaccines
Antonio Cassone
Infectious Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 299, Rome IT 00161,
ITALY, Phone: +39 (0)6 4990 6135, FAX: +39 (0)6 4938 7183, e-mail: cassone@iss.it,
Web: www.iss.it

   Vaccines against human pathogenic fungi , a rather neglected medical need until few years ago,
are now gaining steps in the public health priority scale. The awareness of the rising medical threat
  represented by the opportunistic fungi, particularly among the health care-associated infections ,
     the remarkable advances in the knowledge of fungal pathogenicity, virulence traits and host
 immune response, and the biotechnology progress in the generation of novel immunological tools
   have instilled enthusiasm and new perspectives in the capacity of protecting people from fungal
    diseases by active and passive vaccination. Antibodies have been discovered that may have a
  strong impact in the protection against fungal infection, even beyond the role of antibodies in the
natural history of infection. This discovery has greatly contributed to the advancements in the field
      of fungal vaccines, in recognition that almost all useful vaccines against viral and bacterial
       pathogens owe their protective efficacy to neutralizing, opsonizing or otherwise effective
   antibodies. Overall, there is more hope now than few years ago about the chances of generating
  and having approved by the regulatory authorities one or more antifungal vaccines , be active or
passive, for use in humans in the next few years. In particular, the possibility of protecting against
 multiple opportunistic mycoses in immuno-depressed subjects with a single, well-defined glucan-
  conjugate vaccine, eliciting growth-inhibitory anti-fungal antibodies may be an important step to
                                    achieve this public health goal.




                                                 39
          HOT TOPIC SESSION

SYSTEMS BIOLOGY AND FUNGAL PATHOGENS




                 40
H21
Understanding gene function and drug action
Maureen Hillenmeyer2, Eula Fung2, Sarah Pierce2, Shawn Hoon2, William Lee2,
Michael Proctor2, Robert St. Onge2, Corey Nislow1 and Guri Giaever1
1
  Pharmaceutical Sciences and Medical Genetics, University of Toronto, CCBR 160 College
St., Toronto On M5S3E1, Canada, Phone: 4169787182, FAX: 4169788287, e-
mail: guri.giaever@utoronto.ca, Web: http://chemogenomics.med.utoronto.ca/ 2 Stanford
Genome Technology Center

The identification of drug targets and associated pathways is essential for gaining insight into the
mechanism of drug action and for uncovering potential new pharmaceuticals. The budding yeast
Saccharomyces cerevisiae has proven an effective model system for realizing these two aims.
Because the yeast proteome shares ~50% homology to human compounds that are effective on
yeast proteins they are often effective against their human counterparts. The number of genomic
and proteomic tools for interrogating yeast is vast and growing. For example, complete libraries of
gene knock-outs, over-expression strains, epitope and fluorescently labeled proteins are available.
Such resources, combined with the extraordinary arsenal of genetic tools available provide a
powerful foundation for a high throughput, genome-wide drug discovery effort. Here we
summarize the successes of two methods, haploinsufficiency profiling (HIP) and homozygote
profiling (HOP) aimed at drug target elucidation and elucidating underlying genetic pathways.
Finally we provide a prospective for improving these assays and integrating them with other
systems-levels approaches.




                                                41
H22
Network Analysis of pH Adaptation in Saccharomyces cerevisiae
Darius Armstrong-James, Elaine Bignell, Michael Stumpf and Ken Haynes
Molecular Microbiology & Infection, Imperial College London, Exhibition Road, London
SW7 2AZ, UK, Phone: +44 (0) 20 7594 2072, FAX: +44 (0) 20 8383 3394, e-
mail: k.haynes@imperial.ac.uk

Adaptation to environmental pH is a fundamental process for all microorganisms. Fungal pH
adaptation is particularly important as many fungi are exposed to a wide pH range in the
environment, and internal pH homeostasis is required for many biological functions. Here we use
the core components of the alkaline pH response in Saccharomyces cerevisiae as a biological
exemplar with which to define the relationship between transcriptional programs and growth
phenotypes, in the context of the yeast global interaction network. This allows us to show that
although phenotypic and expression data sets are distinct, they are highly inter-connected and
restricted to a compact region of the yeast interactome. These observations further our
understanding of the network topology of biological processes and provide novel insights into
predicting gene function.




                                              42
H23
Systems Biology of Human Fungal Pathogens; potential and challenges for modeling
Jacky Snoep
Department of Biochemistry, University of Stellenbosch, Private Bag X1, Stellenbosch 7600,
South Africa, Phone: +27218085862, FAX: +27218085863, e-mail: jls@sun.ac.za

The different forms of 'omics research (starting with Genomics) have had a great effect on
development of numerous other fields. The accumulation of large data sets has for instance led to
a boost in Bioinformatics to create software tools for analysis and interpretation. More recently the
importance of having access to complete data sets and the integration of experimental and
modeling approaches has been very important in the field of Systems Biology. My aim in Systems
Biology is to come to a quantitative understanding of the Systems properties in terms of the
characteristics of its components. I have been working mostly on detailed kinetic models of
metabolism and will present some aspects of the construction and validation of such models.
These models have detailed kinetic information on each of the enzyme catalyzed reactions and
mostly focus on the intracellular level. However we have been able to extend one such model on
yeast metabolism to include cell cell interactions. At present the level of detail that can be
included in kinetic models is largely limited by our knowledge of the system. Here one can use
different modeling approaches; for instance structural models can be made without knowledge of
the kinetics in the system and are completely described by the network stoichiometry, which is
known for an increasing number of species and which can be determined largely from the genome
sequence. Kinetic models can be made with different levels of detail, dependent on our knowledge
of the system. In my presentation I will discuss some of the potential applications but also the
challenges of the different modeling approaches with respect to human fungal pathogens.




                                                 43
                   SESSION 4

HOST-FUNGUS INTERACTIONS: THE PATHOGEN ATTACKS…




                      44
S41
Expression of beta-1,2 mannoses at Candida albicans cell wall surface and host damage.
Celine Mille1, Chantal Fradin1, Thierry Jouault1, Pierre André Trinel1, Guilhem Janbon2,
Yann Guerardel3 and Daniel Poulain1
1
  Inserm unit 799 Physiopathologie des Candidoses, Faculty of Medicine/University Hospital,
Place Verdun, Lille 59, FRANCE, Phone: +33 (0)3 2062 3420, FAX: +33 (0)3 2062 3416, e-
mail: dpoulain@univ-lille2.fr 2 Unité de Mycologie Moléculaire. Insitut Pasteur. Paris.
France 3 Glycobiologie Structurale et Fonctionnelle. CNRS 8576 University of Science.
Villeneuve d'Ascq. France;

C. albicans builds mannose blocks bound through an unusual type of linkage: the b-1,2
oligomannosides ( -Mans). Prominent expression of -Mans at the C. albicans cell wall surface is
a phenotypic character contributing to virulence. The unique spatial conformation of -Mans is
specifically detected by mammals innate and adaptative immune systems. -Mans act as adhesins
for host cells, induce specific responses through binding to galectin-3, and TLR2 dependent
pathways. Generation of anti- -Man antibodies confer animal protection against systemic and
mucosal candidosis. These results were obtained by using -Mans released from the cell wall or
chemically synthesized, but information concerning -Man expression at the C. albicans cell wall
and biogenesis is still limited. The presence of -Mans has only been chemically established in
two non-covalently linked molecules: the phosphopeptidomannan (PPM) and the
phospholipomannan (PLM), whereas immunochemical observations suggest that -Man epitopes
may be present in the glycan moiety of cell wall mannoproteins. The genes responsible for -
Mans biogenesis are unknown. These questions were addressed jointly. First, a proteomic-
glycomic analysis of C.albicans cell wall mannoproteins demonstrated that -Mans were probably
present as O-glycans in family members of the so called PIR and GPI anchored cell wall proteins.
Second, we identified the family of genes involved in beta mannose transfer (BMT) in C.
albicans. The construction of individual deletion mutants, combined with immunochemical and
structural analysis, showed that each of the 9 C. albicans genes encodes for Bmtps that are
differentially involved in the sequential steps of -mannosylation of PPM, PLM and
mannoproteins. Surprisingly, individual deletions had only limited impact on “ -Mans surface
antigenic load” and virulence. This is in agreement with previous studies having evidenced a
complex expression mechanism and suggested a possible regulation of -Man expression. In
parallel, immunological studies have shown that the mode of presentation of -Mans to the hosts
by C. albicans, i.e. the nature of the carrier molecule and the length of -Man, chain could control
of the balance between inflammatory and anti-inflammatory responses. As far as all the genes and
all the potential carrier molecules have been identified, it can be anticipated that the questions of
  -Mans functions in C. albicans and its hosts can now be more rationally addressed.




                                                 45
S42
The genetics of the Cryptococcus neoformans capsule
Frédérique Moyrand, Fontaine Thierry and Guilhem Janbon
Mycologie Moléculaire, Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France, Phone: 33
(0)145688356, FAX: 33 (0)145688420, e-mail: janbon@pasteur.fr

The interactions between a pathogen and the infected host are the key to the pathogenesis of many
infections. These involve different types of surfaces molecules and can be proteins, lipids or
polysaccharides. However, most studies focus on proteins because they are easier to eliminate or
modify through gene mutation. Polysaccharides are much more complicated to modify and their
genetics, at least in eukaryotes, is far from being completely understood. However, it is obvious
that, as in bacteria, their structures have a major effect on their function and on the virulence of the
micro-organisms. The cell surface of the pathogenic basidiomycete yeast Cryptococcus
neoformans is mainly composed of polysaccharides. The C. neoformans capsule represents a
fascinating structure and we study the pathways that control its biosynthesis. It primarily
comprised of two polysaccharides: glucuronoxylomannan (GXM, 88% of the capsule mass) and
galactoxylomannan (GalXM, 7% of the capsule mass). We constructed a large collection of
mutant strains in which genes potentially involved in capsule biosynthesis were deleted. We used
a new post-genomic approach to study the virulence of the strains. Primers specific for unique tags
associated with the disruption cassette were used in a real-time PCR virulence assay to measure
the fungal burden of each strain in different organs of mice in multi-infection experiments. With
this very sensitive assay, we identified a putative UDP-glucose epimerase (Uge1p) and a putative
UDP-galactose transporter (Ugt1p) essential for C. neoformans virulence. The uge1 and
ugt1&#61472;strains are temperature sensitive and do not produce GalXM but synthesise a larger
capsule. These mutant strains (GalXM negative, GXM positive) are not able to colonise the brain
even at the first day of infection whereas GXM-negative strains (GalXM positive) can still
colonise the brain, although less efficiently than the wild-type strain.




                                                  46
S43
Small molecule mediation of Aspergillus fumigatus virulence attributes.
Nancy Keller
Plant Pathology and Medical Microbiology Immunology, University of Wisconsin, 1630
Linden Drive, Madison wi 53706, United States, Phone: 608 262-9795, FAX: (608) 263-2626,
e-mail: npk@plantpath.wisc.edu, Web: http://www.plantpath.wisc.edu/fac/npk.htm

Aspergillus fumigatus is the primary causal agent of invasive aspergillosis (IA), a life-threatening
disease associated with immunocompromised patients. The Aspergillus genus is renown for
production of small, bioactive molecules, commonly referred to as secondary metabolites (SM),
thought to yield fitness attributes to the producing species. Recent studies aimed at clarifying
involvement of SMs in IA have uncovered a role for a global regulator of SM production, LaeA,
as well as a role for a class of fatty acid derived signaling SMs known as oxylipins. Our current
understanding of these factors in fungal and disease development will be discussed.




                                                 47
S44
Integrated proteomic and genomic strategies bring new insight into Candida albicans
response upon macrophage interaction
Elena Fernández-Arenas, Virginia Cabezón, Clara Bermejo, Javier Arroyo, César Nombela,
Rosalía Díez-Orejas and Concha Gil
Microbiology, Complutense University, Plaza de Ramón y Cajal s/n, Madrid 28040, Madrid,
Phone: 34-91-3941744, FAX: 34-91-3941745, e-mail: conchagil@farm.ucm.es,
Web: http://www.ucm.es/info/mfar/

The interaction of C. albicans with macrophages is considered a crucial step in the development of
an adequate immune response in systemic candidiasis. The single utility of proteomic and
genomic approaches to study host -pathogen interaction has been previously described in the
literature (1, 2, 3). An in vitro model of phagocytosis that includes a differential staining
procedure to discriminate between internalized and non-internalized yeast was developed. Upon
optimization of a protocol to obtain an enriched population of ingested yeasts, a thorough genomic
and proteomic analysis was carried out on these cells. Both proteins and mRNA were obtained
from the same sample and analyzed in parallel. The combination of 2D-PAGE with MS revealed a
total of 132 differentially expressed yeast protein species upon macrophage interaction. Among
these species, 67 unique proteins were identified. This is the first time that a proteomic approach
has been used to study C. albicans-macrophage interaction. We provide evidence of a rapid
protein response of the fungus to adapt to the new environment inside the phagosome by changing
the expression of proteins belonging to different pathways. The clear down-regulation of the C-
compound metabolism, plus the up-regulation of lipid, fatty acid, glyoxylate and tricarboxylic acid
cycles, indicates that yeast shifts to a starvation mode. There is an important activation of the
degradation and detoxification protein machinery. The complementary genomic approach led us to
detect specific pathways related to Candida’s virulence. Network analyses allowed us generate a
hypothetical model of Candida cell death after macrophage interaction, highlighting the
interconnection between actin cytoskeleton, mitochondria and autophagy in the regulation of
apoptosis (4). In conclusion, the combination of genomic, proteomic and network analyses is a
powerful strategy to better understand the complex host-pathogen interactions. 1-Lorenz MC,
Bender JA, Fink GR. 2004. Eukaryot Cell. 3, 1076 -87. 2-Fradin C, De Groot P, Mac Callum D,
Schaller M, Klis F, Odds FC, Hube B.2005. Mol Microbiol. 56, 397 -415. 3-Martínez-Solano L,
Nombela C, Molero G, and Gil C.2006. Proteomics.1, S133-44.




                                                48
S45
NAD+ and the regulation of the EPA family of adhesins in Candida glabrata
Biao Ma, Renee Domergue, Margaret Zupancic, Shih-Jung Pan and Brendan Cormack
Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe
St., Baltimore MD 21210, USA, Phone: 410 614 44923, FAX: 410 502 6718, e-
mail: bcormack@jhmi.edu

The opportunistic yeast pathogen Candida glabrata adheres to host epithelial cells via a large
family of surface expressed adhesin proteins encoded by EPA genes. To understand the role of
different Epa adhesins in virulence, we are analyzing regulation and ligand specificity for different
members of the EPA family. We have shown that at least 6 of the EPA genes encode adhesins
capable of binding epithelial or endothelial cells and are using glycan microarrays to assess ligand
specificity for these. In analyzing transcriptional regulation of the EPA family, we have
concentrated on the fact that many of the EPA genes are encoded at sub-telomeric loci and are
transcriptionally silenced by chromatin silencing machinery similar to that found in S. cerevisiae.
One particular sub-telomeric adhesin, EPA6 is normally silenced but is expressed during murine
urinary tract infection (UTI). Expression of EPA6 in the urinary tract is due, at least in part, to
limitation for vitamin B3 (niacin, nicotinic acid (NA)) a precursor of NAD+. C. glabrata is a NA
auxotroph, and as a result relies on environmental vitamin precursors as its source of NAD+. In the
simplest model, under limiting environmental NA concentrations, NAD+ levels fall, abrogating
function of the NAD+-dependant histone deacetylase protein Sir2 de-repression of silent chromatin
structure, transcription of the sub-telomeric EPA genes are de-repressed. To further characterize
the role of NA-limitation in regulating gene expression, we have analyzed the role of additional
NAD+-dependent histone deacetylases Hst1 and Hst2. Using microarrays for C. glabrata, we have
identified genes whose transcription responds to NA limitation, and which are regulated either by
Sir2 or Hst1. We have characterized the role of some NA-regulated genes and show that they
function as transporters for various NAD+ precursors. We are currently characterizing the relative
contribution of different Hst1 and Sir2-regulated genes to overall virulence and this data will be
presented.




                                                 49
                    SESSION 5

HOST-FUNGUS INTERACTIONS: … AND THE HOST RESPONDS




                       50
S51
Malassezia sympodialis – host-microbe interactions
Annika Scheynius
Clinical Allergy Research Unit, Dept of Medicine Solna, Karolinska Institutet, Karolinska
University Hospital Solna L2:04, Stockholm 171 76, Sweden, Phone: +46 8 5177 5934,
FAX: +46 8 335724, e-mail: annika.scheynius@ki.se

The yeast Malassezia belongs to the normal cutaneous microflora of human and warm-blooded
animals. The genus comprises today of 11 species and all except for M. pachydermatis require
long-chain fatty acids for growth, why they are most aboundant at sebum-containing skin sites.
Malassezia can cause skin infections, even systemic infections, and is considered as a contributing
factor in atopic eczema (AE), a common chronic inflammatory pruritic skin disease. Pathogenesis
of AE is likely to result from a genetic predisposition, defective skin barrier and environmental
factors such as microorganisms. Elevated serum levels of IgE are frequent in AE patients,
suggesting that allergens play a role in the pathogenic mechanisms. Microorganisms have been
shown to trigger allergic symptoms by acting as adjuvant or even as allergens. One such
microorganism is Malassezia. Specific IgE and/or T-cell reactivity to Malassezia can be detected
in 70 % of adult patients with AE, and antifungal treatment ameliorates the eczema and lowers the
serum IgE levels. We have therefore chosen M. sympodialis, the most common Malassezia
species isolated on skin both from healthy individuals and patients with AE, as a model to deepen
our understanding of how a member of the normal skin flora can interact with the innate and
adaptive immune system and contribute to the pathogenesis of AE. We have demonstrated that
immature human dendritic cells (DCs) can efficiently internalize M. sympodialis and allergenic
components from the yeast in the absence of IgE antibodies, implying that sensitization to M.
sympodialis can be mediated by DCs in the skin. Patients with AE have a higher pH value of the
skin surface. M. sympodialis cultured at this higher pH release more allergens, supporting that the
disturbed skin barrier in AE patients provides an environment that can enhance the production and
release of M. sympodialis allergens into the skin. To date 13 allergens from Malassezia have been
cloned (Schmid-Grendelmeier P et al, 2006 Chem Immunol Allergy. Basel, Karger 91, 98) and the
crystal structure determined for three of them. Interestingly, 4 of the M. sympodialis allergens
show no significant sequence similarity to known proteins, whereas others share similarities with
each other, other yeast proteins or human structures. Thus, cross-reactivity between these allergens
and their human homologues could occur adding the dimension of autoreactivity in the
pathogenesis of AE.




                                                51
S52
Identification of Drosophila Gene Products Required for Phagocytosis of Candida albicans
Shannon L Stroschein-Stevenson, Edan Foley, Patrick H O'Farrell, and Alexander D Johnson.
University of California, San Francisco, California, United States of America.

Phagocytosis is a highly conserved aspect of innate immunity. We used Drosophila melanogaster
S2 cells as a model system to study the phagocytosis of Candida albicans by screening an RNAi
library representing 7,216 fly genes conserved among metazoans. After rescreening the initial
genes identified and eliminating certain classes of housekeeping genes, we identified 184 genes
required for efficient phagocytosis of C. albicans. Diverse biological processes are represented,
with actin cytoskeleton regulation, vesicle transport, signaling, and transcriptional regulation being
prominent. Secondary screens using Escherichia coli and latex beads revealed several genes
specific for C. albicans phagocytosis. Characterization of one of those gene products,
Macroglobulin complement related (Mcr), shows that it is secreted, that it binds specifically to the
surface of C. albicans, and that it promotes its subsequent phagocytosis. Mcr is closely related to
the four Drosophila thioester proteins (Teps), and we investigated these proteins, as well. We
found that TepII is required for efficient phagocytosis of E. coli (but not C. albicans or
Staphylococcus aureus) and that TepIII is required for the efficient phagocytosis of S. aureus (but
not C. albicans or E. coli). Thus, this family of fly proteins distinguishes different pathogens for
subsequent phagocytosis.

Reference: Stroschein-Stevenson et al. (2006) PLoS Bio1 4(1): e4.




                                                 52
S53
Role of Dectin-1 in anti-fungal immunity
Gordon Brown
IIDMM, University of Cape Town, Anzio Road, Cape Town 7925, South Africa, Phone: 27 21
406 6684, FAX: 27 21 406 6029, e-mail: gordon.brown@mweb.co.za,
Web: http://www.iidmm.uct.ac.za/gbrown/

The innate ability to detect pathogens is essential for multicellular existence, and has been
achieved through the evolution of germ-line encoded receptors which can recognise non-self
structures, the so-called “pattern recognition receptors” (PRR). The structures recognised by these
receptors, termed the pathogen associated molecular patterns (or PAMPs), trigger responses
designed to protect the host from the invading pathogen, forming part of the innate immune
system found in all higher organisms. The innate recognition of fungi, in particular, has been
attributed to various opsonic and non-opsonic PRRs which recognise a range of fungal PAMPs,
including beta-glucan; a carbohydrate polymer which can comprise up to 50% of the fungal cell
wal. A number of receptors for beta-glucans have been identified, but only Dectin-1 has been
clearly demonstrated to play a role in mediating cellular responses to these carbohydrates. Dectin-
1 is a type II transmembrane glycoprotein with a single extracellular non-classical C-type
carbohydrate recognition domain (CRD) and a cytoplasmic tail possessing an immunoreceptor
tyrosine-based activation-like (ITAM) motif. Dectin-1 is predominantly expressed on myeloid
cells and recognises soluble and particulate beta glucans, as well as an unidentified ligand on T-
cells. We and others have demonstrated that this receptor mediates a variety of cellular responses
to beta-glucans, including phagocytosis, endocytosis and the oxidative burst and can induce the
production of arachidonic acid and numerous cytokines and chemokines. These responses are
triggered through the cytoplasmic ITAM-like motif of this receptor, utilising novel signalling
pathways involving a unique interaction with Syk kinase and collaborative signalling with the
TLRs. We have shown that Dectin-1 can recognise several fungal species, including Candida
spp., Pneumocystis spp., Saccharomyces spp., Coccidoides spp. and Aspergillus spp.. We and
others have shown that Dectin-1 contributes to the uptake and killing of live fungal particles, in
part through the induction of the respiratory burst, and the production of cytokines and
chemokines. We have recently generated a mouse deficient for this receptor, and have shown that
these animals have an enhanced susceptibility to fungal infection. These results therefore establish
a fundamental role for Dectin-1 in anti-fungal immunity and represent the first demonstration of a
signalling non-Toll-like pattern recognition receptor required for the induction of protective
immune responses.




                                                53
S54
Purified capsular material of C. neoformans determines immunosuppression by direct
engagement to Fc gamma RIIB receptor
Anna Vecchiarelli
Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto,
Perugia 06126, ITALY, Phone: +39 075 585 7407, FAX: +39 075 585 7407, e-
mail: vecchiar@unipg.it

The capsule of C. neoformans is generally considered to be the most important virulence factor
and contributes to virulence by inhibiting phagocytosis and interfering with an effective immune
response. Glucuronoxylomannan (GXM) is the major polysaccharide component in the capsule.
GXM has profound effects on both innate and adaptive immunity mechanisms. In this study we
demonstrated that GXM exerts potent immunosuppression by direct engagement to
immunoinhibitory receptor FcgammaRIIB. Activation of FcgammaRIIB by GXM leads to the
recruitment and phosphorylation of SHIP that prevents IkappaBalpha activation. The
FcgammaRIIB blockade inhibits GXM-induced IL-10 production and induces TNF-alpha
secretion. GXM quenches LPS-induced TNF-alpha release via FcgammaRIIB. The addition of
monoclonal antibody to GXM reverses GXM–induced immunosuppression by shifting recognition
from FcgammaRIIB to FcgammaRIIA. These findings indicate a novel mechanism by which
microbial products can impair immune function through direct stimulation of an inhibitory
receptor. Furthermore, our observations provide a new mechanism for the ability of specific
antibody to reverse the immune inhibitory effects of certain microbial products.




                                              54
S55
Immunity to Cryptococcus neoformans: O-Man, that’s sweet
Stuart Levitz
Infectious Diseases, University of Massachusetts, 364 Plantation Street, Worcester MA 01605,
USA, Phone: 508-856-1525, FAX: 5088561828, e-mail: stuart.levitz@umassmed.edu,
Web: http://www.umassmed.edu/ivp/faculty/levitz.cfm

C. neoformans is a major pathogen in persons with deficient T-cell immunity. The
glucuronoxylomannan capsule confers virulence to the fungus. My laboratory has been studying
C. neoformans mannoproteins (MP), a heterogeneous group of antigens that elicit cell-mediated
immune responses in mice and humans. MP share a C-terminal serine/threonine (S/T) rich region,
that is the site of heavy O-linked mannosylation, followed by a glycosylphosphatidylinositol (GPI)
anchor that presumably serves as a cell wall attachment site. We have hypothesized that the
extensive mannosylation plays an essential role in immune stimulation by targeting MP to
mannose receptors (MR) on antigen-presenting cells (APC). Two MR, the macrophage mannose
receptor (MMR) and dendritic cell-specific ICAM-3-grabing nonintegrin (DC-SIGN), were shown
to bind MP. Conversely, MR blockade with mannosylated ligands reduced uptake of MP and
inhibited T-cell activation. The immunodominant APC responsible for immune stimulation was
shown to be dendritic cells (DC). The kinetics of native and recombinant MP capture by DC were
rapid and dependent on MR. By confocal microscopy, intracellular MP co-localized with MHCII,
MMR and DC-SIGN. However, all is not rosy as MP neither stimulates IL-12p70 release nor
upregulates the maturation markers MHCII, CD40, CD80 and CD86. Moreover, MP elicits only
partial protection when administered to mice prior to challenge with live C. neoformans. Studies
are in progress examining methods of boosting and skewing immune responses by administering
MP combined with adjuvants which stimulate TLR and dectin-1. These studies suggest that DC
provide the crucial link between innate and adaptive immune responses to C. neoformans via a
process by which MR efficiently bind and internalize MP. However, optimal antigen presentation
and the subsequent initiation of a protective T cell response appear to require additional stimuli.




                                                55
        ABSTRACTS

POSTERS AND WORKSHOP TALKS




            56
P1A
Phylogenetic Diversity of Fungal Osmotic and Cell Wall Stress-Signalling Proteins
Elissavet Nikolaou and Alistair Brown
Molecular and Cell Biology, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD,
UK, Phone: +44-1224-555888, FAX: +44-1224-555844, e-mail: e.nikolaou@abdn.ac.uk,
Web: www.abdn.ac.uk

Stress responses are required for survival of microbes and pathogenic microbes in their host due to
their highly variable environment. Cells sense changes in their environment (detection of external
signals) and through signal transduction pathways (information routes) trigger an appropriate
response [1, 2]. Saccharomyces cerevisiae has been chosen as a model organism in this study, as
individual signalling pathways have been studied indetail in this organism using combination of
genetical, molecular, biochemical and genomic tools [3, 4]. Comparative genomic analysis from
our laboratory is revealing evolutionary differences in the osmotic [3] and cell wall [5] stress
responses of pathogenic and benign fungi (e.g. Saccharomyces cerevisiae, Candida albicans and
Schizosaccharomyces pombe). This study is examining the differences in the "Relative
Conservation Indeces", according to corresponding functional peer groups of Saccharomyces
cerevisiae genome proteins, of evolution of osmotic as well as cell wall stress signalling molecules
across benign and pathogenic fungi, which have had their genomes sequenced. To date we have
generated a robust phylogenetic tree for the 18 sequenced fungi under investigation and compared
the degree of conservation for osmotic and cell wall stress signalling molecules. The implications
of the data will be discussed.
1. Chen et al. (2003). Global transcriptional responses of fission yeast to environmentalstress. Mol
Biol Cell. 14, 214-229.
2. Hohmann, S. (2002). Osmotic stress signalling and osmodaptation in yeasts.Microbiol Mol Biol
Rev. 66, 300-372.
3. Krantz et al. (2006). Comparative genomics of the HOG-signalling system in fungi.Current
Genetics 49, 137-151.
4. Krantz et al. (2006). Comparative analysis of HOG pathway proteins to generatehypotheses for
functional analysis. Current Genetics 49, 152-165.
5. Levin, D. (2005). Cell wall integrity signalling in Saccharomyces cerevisiae.Microbiology and
Molecular Biology Reviews. 69, 262-291.




                                                57
P2B
Micro-variation in multiple and single isolation sets of Candida albicans
Mette D. Jacobsen1, Judith M. Bain1, Amanda D. Davidson1, Arianna Tavanti2, David
H. Ellis3, Chris C. Kibbler4, Duncan J. Shaw1, Neil. A.R. Gow1 and Frank C. Odds1
1
  Aberdeen Fungal Group, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK,
Phone: +44 1224 555888, FAX: +44 1224 555844, e-mail: m.d.jacobsen@abdn.ac.uk2
Department of Biology, University of Pisa, Pisa, Italy3 Department of Microbiology and
Immunology, Australian Candidaemia Study, Women’s and Children’s Hospital, North
Adelaide, SA, Australia4 Department of Medical Microbiology, Royal Free and University
College Medical School, Royal Free Hospital, London, UK

One important tool in understanding the epidemiology and phylogenetics of Candida albicans as
commensal and pathogen is multi-locus sequence typing (MLST) where isolates are typed based
on the variability of nucleotide sequences in a number of housekeeping genes.As a part of our
ongoing analysis of C. albicans population structure by MLST we hold sets of multiple isolates
from several individual patients and healthy volunteers. We studied 59 sets of multiple isolates
including sequential isolates from patients receiving antifungal treatment, sets of isolates from
superficial and sterile sites from the same patient, isolates maintained and passaged through
different culture conditions, isolates experimentally exposed to antifungals and commensal
isolates from healthy carriers. In addition to MLST, isolates were typed by ABC typing of rRNA
genes and the homozygosity or heterozygosity at the mating-type-like locus (MTL) was
determined.A high proportion (49/59; 83%) of sets of isolates showed indistinguishable or closely
related strain types by both MLST and ABC typing. Microvariation, evidenced as small
differences between MLST types in a set, resulted in most instances from a loss of heterozygosity
(LOH) at one or more of the sequenced loci. Two cases of strain replacement were observed
where separate isolates from a patient varied in both MLST and ABC type at different hospital
admissions.To examine possible strain variability in individual isolations, six colonies were typed
from each primary isolation plate for oral samples from five C. albicans-positive healthy
volunteers and four C. albicans-positive vaginal samples. Among these nine sets of 6 colonies
typed, one showed two distinctly different MLST types, five showed LOH-based variations
between colonies and only three gave sets of indistinguishable colonies. We conclude that LOH
variability in C. albicans genes is a common event, even within colonizing and infecting
populations, and may serve as a substitute for sexually achieved diversity.




                                                58
P3C
Histoplasma capsulatum and Pneumocystis spp. co-infection in wild bats in Latin America
Laura Elena Carreto-Binaghi1, Maria Lucia Taylor1, Gabriela Rodriguez-Arellanes1, El
Moukhart Aliouat2, Cécile- Marie Aliouat2, Eduardo Dei-Cas2 and Magali Chabé2
1
  Lab. Inmunología de Hongos,Dept. Microbiología-Parasitología, Facultad Medicina,
Universidad Nacional Autónoma de México, Universidad 3000, Circuito escolar, Ciudad
Universitaria, Mexico 04510, MEXICO, Phone: +52 (55) 5623-2462, FAX: +52 (55) 5623-
2462, e-mail: lauraelena_c@yahoo.com, Web: www.histoplas-mex.unam.mx 2 Faculté des
Sciences Pharmaceutiques et Biologique, Université de Lille-2, Lille, 3 rue du Pr Laguesse- BP
87-59 006 Lille cedex

Histoplasmosis due to Histoplasma capsulatum var. capsulatum (HHC) and pneumonia caused by
Pneumocystis spp. are systemic fungal diseases that could develop in susceptible hosts. Clinical
presentations vary from mild-to-severe, mainly in immunocompromised individuals. These
microorganisms cause host infection through respiratory airway, affecting lung tissue and
disseminating to other organs. Etiologic agents have a wide genetic diversity, which justify their
study on epidemiologic, taxonomic, and systematic bases. Pneumocystis’ species in America’s
mammals have been explored scarcely. In Europe, Pneumocystis-host specificity has been
determined either pheno- or genotypically, and the characterization of these organisms in
symbiosis with wild animals remains as an important aim to understand this pathogen
dissemination in nature. H. c. capsulatum is endemic in USA and many Latin American countries.
People who work in bats’ habitat have an increased risk of acquiring HHC. Recently, co-infected
bats with H. c. capsulatum and Pneumocystis spp. have been found, involving these mammals as a
probable reservoir and disperser of both parasites. To evaluate the impact of H. c. capsulatum and
Pneumocystis spp. infections in wild bats in Latin America, lung samples obtained from randomly
captured bats were monitored, using molecular techniques. Fifty-four bats’ lungs from Mexico and
eleven from French Guyana (FG) were screened for H. c. capsulatum using nested-PCR with
Hcp100 protein coding gene and for Pneumocystis infection using the mtLSUrRNA sequence
analyses. Out of nine Histoplasma positive samples, one from FG bats revealed co-infection with
Pneumocystis. According to the genetic distance analysis based on Hcp100 sequences, differences
between the two Latin American groups studied were detected. The present research is being
supported by the grants: SEP-CONACYT-ANUIES-ECOS-M05-A03 (Mexico-France) and
DGAPA-UNAM-IN203407-3. Bialek R, et al., (2002), J Clin Microbiol, 40, 1644. Demanche C,
et al., (2001), J Clin Microbiol, 39, 2126. Taylor ML, et al., (2005), FEMS Immunol Med
Microbiol, 45, 451.




                                               59
P4A
Candida albicans orthologues of yeast meiotic genes have diverse functions in Candida
biology.
Ute Zeidler, Michael Breitenbach and Arnold Bito
Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, Salzburg 5020,
Austria, Phone: +43 662 8044 5793, FAX: +43 662 8044 144, e-mail: ute.zeidler@sbg.ac.at

Although it has been shown that Candida albicans cells of opposite mating type are able to mate,
meiosis of neither the tetraploid mating products nor of diploid cells has never been observed.
Recently we have shown that the Candida orthologoues of the DIT1 and DIT2 genes of S.
cerevisiae, which are expressed exclusively during yeast meiosis, are able to complement
functionally the defects of the corresponding yeast mutant strains. Although Candida null mutants
did not show any phenotype when grown in vitro, they behaved significantly less virulent
compared to the corresponding reconstituted strains and the wild-type strain in a mouse model of
systemic infection.In the present study we studied a set of 14 further Candida orthologues of yeast
genes that are needed for meiotic and ascospore development. Homozygous null mutants and
reconstituted strains for each gene were constructed and the phenotype was studied under several
different growth conditions and in the presence or absence of metabolic inhibitors. The expression
of the genes was investigated in yeast and hyphal growth.For seven genes we could detect a
mutant phenotype. Two genes, CAK1 and IME2, both encoding protein kinases, appear to be
essential for growth in C. albicans. The null mutants of two other Candida genes, NDT80 and
UME6, which encode transcriptional regulators, were unable to form true hyphal filaments upon
induction of hyphal development. The null mutants of four genes, NDT80, RIM101, SPO73 and
GCS1, which encode for different types of proteins, showed a significantly higher susceptibility to
several toxic compounds. Each of these genes appears to be involved either or both in oxidative
stress response and/or in resistance to drugs currently used in antifungal therapy. The gene
expression analysis revealed that most of the genes are expressed under standard in vitro growth
conditions. The UME6 transcript was detectable only in hyphal cells and not in the yeast cells.
This confirms the suggestion indicated by the mutant study, that CaUME6 is involved in the
regulation of hyphal development.These results suggest that the orthologues of yeast meiotic
genes have adopted diverse functions in C. albicans that are not related to meiotic development.In
the near future the phenotypic analysis of the mutant strains will be further investigated under
growth conditions not yet studied and complemented by gene-specific experiments. These and the
results mentioned above will be presented.




                                                60
P5B
Transcriptional and physiological adaptation to defective protein-O-mannosylation in
Candida albicans
Pilar D. Cantero1, Christian Lengsfeld2, Stephan K.-H. Prill2, Marina Subanovic2,
Elvira Román3, Jesús Pla3 and Joachim F. Ernst2
1
  Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de
Unamuno, Salamanca 37007, Spain, Phone: +34 923 294677, FAX: +34 923 224876, e-
mail: soile@usal.es2 Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf,
Germany.3 Departamento de Microbiología II, Facultad de Farmacia, Universidad
Complutense de Madrid, Spain.

Five Pmt isoforms O-mannosylate secretory proteins in Candida albicans. Comparisons of
genome-wide transcript patterns of each pmt mutant revealed commonly downregulated genes
involved in glycolysis and glycerol production. Increased phosphorylation of the Cek1p- but not
the Mkc1p-MAP kinase, as well as increased transcript levels for stress-related genes were
detected in the pmt1 strain but not in the other pmt mutants. The transcriptomal pattern after short
term-inhibition of Pmt1p-activity confirmed such stress responses, but did not indicate an
alteration of glycolytic flow. Short- but not long-term adaptation to Pmt1p inhibition required
signaling components Cek1p, Mkc1p, Efg1p and Tpk1p, while lack of Cna1p (calcineurin) was
essential for survival during Pmt1p-inhibition; accordingly, cyclosporin A strongly inhibited
growth of the pmt1 mutant. The lack of Pmt isoforms influenced transcript levels for the
remaining isoforms both positively and negatively, suggesting complex cross-regulation among
PMT genes. These results confirm individual functions of Pmt isoforms but indicate also a
common biphasic adaptation response to Pmt deficiency. While known signaling pathways
modulate or, in the case of calcineurin, are essential for short-term adaptation, long-term
adaptation likely occurs independently of stress pathways but may require adjustments of
remaining Pmt activities and of glycolytic flow.




                                                61
P6C
Candida albicans CHT4 encodes a sporulation specific gene
Alex Dünkler2 and Jürgen Wendland1
1
  Yeast Biology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen 2000, Denmark,
Phone: +45 3327 5230, FAX: +45 3327 4708, e-mail: jww@crc.dk2 Leibniz Institute for
Natural Products Research and Infection Biology - Hans Knöll Institute and Friedrich Schiller
University, Jena

The C. albicans genome encodes four chitinases, CHT1, CHT2, CHT3, and CHT4. In a recent
analysis we have shown that Cht3 encodes the functional homolog of the Saccharomyces
cerevisiae chitinase Cts1 that is involved in mother-daughter cell separation. All four chitinase
encoding genes are non-essential. They belong to two groups in which CHT1, CHT2, and CHT3
are more similar to CTS1, while CHT4 is more similar to CTS2. ScCTS2 was described as a
“sporulation specific gene”. However, published experimental evidence does not exist. The
diploid cts2/cts2 strain sporulates well and the spores do not exhibit germination defects or
increased sensitivity against zymolyase. In the filamentous fungus Ashbya gossypii a CTS2
homolog (ACL166w) was identified. The AgCts2 is 490aa in length and shows 42.3% overall
identity to ScCts2 (511aa) and 33.2% identity to CaCht4 (388aa). The catalytic centers of the three
proteins are highly conserved and shows clear differences to members of the Cts1-chitinase
family. We deleted the AgCTS2 gene and carried out phenotypic characterizations. The Agcts2
mutants showed no growth retardation or morphogenetic defects. However, Agcts2 mutants
revealed a spore shape defect indicating defects in the assembly of the spore wall. This defect did
not lead to increased sensitivity against zymolyase or heat shock and did not affect the
germination pattern of A. gossypii spores. Expression of AgCTS2 was analyzed using a lacZ
reporter gene. AgCTS2 is expressed in the center of a mycelium that corresponds to the
sporogenous mycelium.The spore wall defect of Agcts2 mutants could be complemented by either
AgCTS2 or the S. cerevisiae CTS2, and the C. albicans CHT4 gene. Expression of ScCTS2 or
CHT4 was achieved by either the AgCTS2 or the AgTEF1 promoter. Therefore, these data
indicate that C. albicans CHT4 encodes a fully functional chitinase that is specific for spore wall
assembly further fuelling efforts to elucidate a complete sexual cycle for C. albicans.




                                                62
P7A
A unique CUG codon distribution in the Candida albicans genome
Isabel Miranda1, Rita Rocha2, Denisa Mateus1, Hugo Pais3, Miguel Pinheiro3, Sandra Macedo-
Ribeiro4 and Manuel A.S. Santos1
1
  Biology and CESAM, University of Aveiro, Campus de Santiago, Aveiro 3810-193,
PORTUGAL, Phone: +351 234370970, FAX: +351 234426408, e-mail: imiranda@bio.ua.pt2
Center for Neurosciences and Cell Biology, University of Coimbra, Portugal3 IEETA,
University of Aveiro, Portugal4 IBMC, University of Oporto, Portugal

Most organisms use the standard genetic code, however several alterations have been found in
prokaryotes and eukaryotes, invalidating the hypothesis that the genetic code is universal and
frozen. Most alterations occur in mithocondria and the only known case of a cytoplasmatic sense-
to-sense codon identity change occurs in several species of the genus Candida. In the human
pathogen Candida albicans, a novel tRNA(CAG) decodes the standard-CUG codon mainly as
serine (97%) but to a lesser extent as leucine (3%). This dramatic genetic event appeared 272
million years ago and evolved gradually through an ambiguous codon decoding mechanism that
forced the disappearance of approximately 30,000 CUG codons in the Candida ancestor and the
emergence of 13074 “new ambiguous” CUG codons. We have increased CUG ambiguity through
tRNA engineering methodologies to uncover putative roles of such ambiguity. Surprisingly, C.
albicans tolerated up to 30% of CUG ambiguity and such high level of codon mistranslation did
not have any visible impact on the expression of molecular chaperones, proteassome activity or in
glycogen and trehalose accumulation. Thus, CUG ambiguity is not a stressful event to C. albicans,
even at very high levels. To understand this phenotype at the molecular level, we have carried out
an analysis of multiple sequence alignments and protein 3D-models, which revealed that most
CUG-encoded residues are located in non-conserved regions of protein structure and are partially
exposed to the solvent in positions where both hydrophobic leucine and polar serine could be
accommodated without disrupting protein structure. This data showed that C. albicans has a
unique genome distribution of CUG codons minimizing proteome disruption.IM is supported by
FCT PosDoc Grant (SFRH/BPD/20842/2004). RR is supported by FCT PhD grant
(BD/15233/2004), the Doctoral Programme on Experimental Biology and Biomedicine,
University of Coimbra and by FEDER/FCT project Nº POCI/BIA-PRO/55472/2004.




                                               63
P8B
Fungal strains isolated from patients in surgery and internal wards of central clinical
hospital of medical university in Warsaw in 2006.
Ewa Swoboda-Kopec,        Maria Dabkowska,      Joanna Kadzielska,     Beata Sulik-Tyszka,
Ewa Stelmach, Dariusz Kawecki, Anna Majewska and Miroslaw Luczak
General Surgery and Transplantation / Medical Microbiology, Medical University, Chalubinski
5, Warsaw 02-004, Poland, Phone: +48 022 622-00-28, FAX: +48 022 628-27-39, e-
mail: dkawecki@o2.pl, Web: www.am.edu.pl

Objective:The aim of this study is to assess the occurrence of fungal strains in patients
hospitalised in surgical and internal wards of Central Clinical Hospital of Warsaw in from January
to December of 2006.

Methods: Mycological tests were performed from 10 632 clinical specimens. All specimens were
inoculated on Sabourauds supplemented with gentamycine and chloramphenicol (Becton
Dickinson). Isolated tribes were identified by using chromagar candida and automatic test ID32C
(Biomerieux). The susceptibility tests to antifungal agents were performed using E-test.

Results: Material elaboration resulted in isolation of 1559 clinical specimens positive in mycology
laboratory examination. In total 1863 strains of yeast-like and moudls were cultured. There were
1800 strains of yeast-like (96,6%) and 63 strains of moudls (3,4%) isolated. Most common fungal
strains cultured were: C. albicans n=809 (43,4%), C. glabrata n=481 (25,8%), C. tropicalis n=117
(6,3%), C.krusei n=114 (6,1%), C. parapsilosis n=96 (5,2%), other fungal strains n=183 (9,8%).

Conclusion 1.Candida albicans was the most common yeast isolated from clinical specimens.
2.Aspergillus spp. and Fusarium spp. were dominated moudls isolated from surgery and internal
hospital wards.




                                                64
P9C
Accelerated loss and fragmentation of chromosome 5 are major events linked to the
adaptation of rad52-deltha strains of Candida albicans to sorbose
Toni Ciudad1,       Encarnación Andaluz1,     Jonathan Gómez-Raja1,  Belén Hermosa1,
Elena Rustchenko2, Richard Calderone3 and Germán Larriba1
1
  Departamento de Microbiología, Universidad de Extremadura, Avda. Elvas s/n, Badajoz
06006, Spain, Phone: +34 924289428, FAX: +34 924289428, e-mail: aciudad@unex.es2
Department of Biochemistry and Biophysics. University of Rochester Medical Center.
Rochester. NY 14642. USA.3 Department of Microbiology and Immunology. Georgetown
University. School of Medicine. Washington DC 20007. USA

C. albicans can adapt and grow on sorbose plates by losing one copy of Chr5. Since rad52
mutants of Saccharomyces cerevisiae lose chromosomes at a high rate, we have investigated the
ability of C. albicans rad52-deltha to adapt to sorbose. Carad52-deltha mutants generate Sou+
strains earlier than wild type but the final yield is lower, probably because they die at a higher rate
in sorbose. As other strains of C. albicans, CAF2 and rad52-deltha derivatives generate Sou+
strains by a loss of one copy of Chr5 about 75% of the time. In addition, rad52 strains were able to
produce Sou+ strains by a fragmentation/deletion event in one copy of Chr5, consisting of loss of
a region adjacent to the right telomere. Finally, both CAF2 and rad52-deltha produced Sou+
strains with two apparent full copies of Chr5, suggesting that additional genomic changes may
also regulate adaptation to sorbose.




                                                  65
P10A
Creation of a Candida lusitaniae congenic strain pair and characterization of the mating
type loci of Candida lusitaniae and Candida guilliermondii
Jennifer L. Reedy1, Laura Y. McGirt2, Christina M. Hull3 and Joseph Heitman1
1
  Molecular Genetics and Microbiology, Duke University, 320 CARL Bldg, Research Drive,
Box 3546, Durham NC 27707, USA, Phone: +001 919 684 3036, FAX: +001 919 684 5458, e-
mail: reedy004@mc.duke.edu 2 Department of Dermatology, The Johns Hopkins Hospital, 601
N. caroline Street, 6th floor, Baltimore, MD 21287 3 Department of Biomolecular Chemistry
and Medical Microbiology and Immunology, University of Wisconsin-Madison School of
Medicine and Public Health, 587 Medical Science Center, 1300 University Avenue, Madison,
WI 53706-1532

Candida lusitaniae and Candida guilliermondii are human pathogenic members of the genus
Candida that possess complete sexual cycles. Both are haploid, dimorphic fungi that are
causative agents of human candidal blood stream infections. Understanding the life cycles of
these Candida species is important in developing a complete understanding of these pathogenic
fungi. Thus far, the majority of effort has been focused on Candida albicans; however, the lack
of a complete meiotic cycle has limited the application of classical genetics for this species.
Developing sexual models in species closely related to C. albicans can provide information
regarding the evolution of the signaling pathways that govern sexual reproduction and also
provide systems for heterologous expression studies and for comparison with C. albicans. To
study complex phenomena in these organisms it is necessary to first develop genetic tools.
Through a series of 12 backcrosses, a congenic pair of a and alpha C. lusitaniae strains that
differ only at the mating type (MAT) locus were generated. The parental strains differed with
respect to filamentation, chromosomal length polymorphisms dictated by PFGE, and
sensitivity to various stresses, whereas the congenic strain pair behaved identically with respect
to these phenotypic and genotypic properties. The structure of the MAT locus was elucidated
in C. guilliermondii and C. lusitaniae to facilitate genetic studies and to understand the
evolution of MAT in the Candida species complex. The C. lusitaniae sequenced strain is alpha
and the parent of the congenic strain pair, thus enabling MATalpha to be readily annotated.
Likewise, the C. guilliermondii MATa allele was annotated as a consequence of the C.
guilliermondii genome project. The opposite MAT locus for each species was identified using
degenerate PCR and sequenced. Similarly to C. albicans, the C. guilliermondii and C.
lusitaniae MAT loci also contain the genes encoding poly A polymerase (PAP1), oxysterol
binding protein (OBP1), and phosphoinositol kinase (PIK1). In C. albicans, the MATalpha
equivalent, MTLalpha, encodes the transcription factors alpha1 and alpha2, and the MATa
equivalent, MTLa, encodes a1 and a2. Interestingly, the C. lusitaniae and C. guilliermondii
MAT loci do not encode the full complement of transcription factors present in C. albicans.
These results have interesting implications regarding the determination of cell identity and the
control of meiosis within these sexual species.




                                                 66
P11B
An Universal Approach for the Analysis of Differential Gene Expression in Fungal
Pathogens
Elena Lindemann1, Bettina Rohde2, Johannes Regenbogen2, Steffen Rupp1 and Kai Sohn1
1
  MBT, Fraunhofer IGB, Nobelstr. 12, Stuttgart 70569, Germany, Phone: +49 711 970 4055,
FAX: +49 711 970 4200, e-mail: kai.sohn@igb.fraunhofer.de2 GATC, Jakob-Stadler-Platz 7,
78467 Konstanz, Germany

The DNA-microarray hybridization technology has emerged as the application of choice for the
identification of differentially expressed genes. Due to the automation and miniaturization, this
method allows a parallel determination of relative transcript abundances for thousands of genes in
a single experiment, but also shows some major limitations. Most importantly, the generation of
microarrays require the availability of completely sequenced and annotated genomes thus limiting
the number of organisms that can be analyzed.Here, we describe a novel approach for global
transcriptional profiling that circumvents the need for any genome sequence information, making
this system universally applicable for any eukaryotic organism. The principle underlying this
novel technique is based on the high-resolution, electrophoretic separation of a complex cDNA
sample in a two-dimensional gel system. Starting with total RNA, double-stranded cDNA is
synthesized and 3’-terminal DNA fragments are selectively amplified by PCR. These are
subsequently separated on the basis of two different criteria: in the first dimension, using non-
denaturing polyacrylamide gel, according to their molecular weight, and in the second dimension
corresponding to the fragments’ GC content, by use of denaturing gradient gel electrophoresis
[DGGE].2D-cDNA-gels are able to resolve more than 2.000 different spots per gel which, in
combination with a selective amplification of subpopulation of ds-cDNA-fragments using
anchored primers, can be used to map a complete eukaryotic transcriptome. Not only the high
resolution of the gel, but also the high level of reproducibility allow a qualitative as well as a
quantitative analysis of spot patterns derived from different samples, which can be used for the
identification of transcripts with significant changes in abundance. The evaluation of
transcriptome data on Candida albicans reveals a high correlation with corresponding DNA-
microarray data, indicating that there is no bias introduced during the PCR amplification of the
cDNA-fragments. Surprisingly, we could also identify some small transcripts, which due to their
small size (< 100 bp), are still not annotated and thus could not be analysed using
microarrays.This straightforward approach was validated with the Candida albicans
transcriptome, but it will be applied to other fungal pathogens like Candida dubliniensis, for
which the annotation of the genome sequence is still in progress.




                                               67
P12C
Characterization of fungal cell wall redox enzymes in the lichen Collema
Ntombizamatshali Mtshali and Richard Beckett
Biology, University of Kwa-Zulu Natal, 22 Carbis Road, Pietermaritzburg 2301, South Africa,
Phone: 44(0) 115 9709398, FAX: 44 (0) 115 3251, e-mail: 204507919@ukzn.ac.za,
Web: www.ukzn.ac.za

Laccases, tyrosinases and peroxidases are oxido-reductase or “redox” enzymes that occur in a
wide range of fungi, animals and plants. These enzymes have been used in numerous
biotechnological applications such as paper bleaching, wine clarification and the bioremediation
of compounds such as trichlorophenol, alkenes and herbicides. In addition, oxidation of various
compounds by these enzymes can results in formation of valuable pharmacological products.
Discovery of new isoforms could further extend the useful applications of these enzymes. The aim
of our current studies is to investigate the occurrence of redox enzymes in lichen-forming fungi.
Using a cell fractionation technique we were able to clearly confirm the presence of tyrosinases,
peroxidases and laccases in the lichen genus Collema. SDS PAGE was used in a preliminary
micro-characterization of the enzymes. Results indicated that the lichen contained single isoforms
of laccase and tyrosinases with molecular masses of c. 400 kDa and 60 kDa respectively. The
lichen also contained two main peroxidase isoforms, with molecular masses of c. 40 kDa. These
enzymes were studied in an extremely desiccation tolerant organism, and may therefore be
suitable for industrial applications.




                                               68
P13A
Using comparative genomics to predict function: recent applications and perspectives for
elucidating pathways involved in pathogenesis.
Toni Gabaldón
Bioinformatics, CIPF, Autopista del Saler, 16, Valencia 46013, SPAIN, Phone: +34 96 328
9680     (ext     1006),      FAX: +34   96     328    9701,    e-mail: tgabaldon@cipf.es,
Web: http://bioinfo.cipf.es/tgabaldon

The genomics era has inspired the development of novel computational methods to characterize
biological processes. In particular, several methods exploit the co-evolution of functionally
interacting proteins others compare the positions of the genes along the chromosomes. We used
these techniques to elucidate biochemical pathways in the mitochondrion and to discover new
disease genes associated with mitochondrial dysfunction. The use of fungal genomes has great
advantages because this group shows a great diversity in their mitochondrial metabolism, which
reflects functional adaptation to various environments. This adaptation has been modulated by
lineage-specific losses and gains of proteins and pathways. The availability of fully sequenced
genomes of several fungal species as well as recent experimental characterization of the
mitochondrial proteome in Saccharomyces cerevisiae and other species allows us to trace the
evolution of the mitochondrial metabolism in this versatile phylum. Proteins functioning in the
same biochemical pathway tend to have a similar history of gene loss events, and we use this
property to predict functional interactions among proteins in our set. A especial emphasis is put on
clinically-relevant proteins from the respiratory chain such as NADH:Ubiquinone oxidoreductase,
which have been lost independently from several fungal species. I will also consider the future
prospects that this new technique opens in the context of the elucidation of pathways involved in
pathogenesis and virulence of human fungal pathogens.




                                                69
P14B
Regulation of growth and development in Aspergillus fumigatus and A. nidulans
Jae-Hyung Mah and Jae-Hyuk Yu
Bacteriology, University of Wisconsin, 1925 Willow Dr., Madison WI 53706, USA, Phone: 1-
608-262-4696, FAX: 1-608-263-1114, e-mail: jyu1@wisc.edu,
Web: http://www.wisc.edu/fri/jyu.htm

The opportunistic human pathogen Aspergillus fumigatus reproduces asxeually by forming a
large number of small conidia. We studied the regulation of asexual development (conidiation)
in A. fumigatus via examining functions of four key controllers, GpaA (G alpha), AfFlbA
(RGS), AfFluG and AfBrlA. Expression analyses of gpaA, AfflbA, AffluG, AfbrlA and
AfwetA revealed that, while transcripts of AfflbA and AffluG accumulate constantly, AfbrlA
and AfwetA are specifically expressed during conidiation. Loss of function AfflbA and
dominant activating GpaAQ204L mutations both cause reduced conidiation coupled with
increased hyphal mass, indicating that GpaA mediates signaling that activates vegetative
growth while inhibiting conidiation. As GpaA is the primary Galpha for AfFlbA function, the
dominant interfering GpaAG203R mutation suppressed the phenotype resulting from loss of
AfflbA function. These results corroborate the idea that the principal roles of G proteins and
RGSs are conserved in aspergilli. Functions of the two major developmental activators AfFluG
and AfBrlA are then examined. Whereas deletion of AfbrlA eliminated conidiation completely,
deletion of AffluG did not result in severe defects in A. fumigatus sporulation in air-exposed
culture. These results imply that, while the two Aspergillus species may have a common key
downstream developmental activator, upstream mechanisms activating brlA may be different.
Finally, both AffluG and AfflbA mutants showed reduced conidiation and delayed
accumulation of AfbrlA mRNA in developmentally induced cultures, indicating that these
upstream regulators are associated with the proper progression of conidiation.




                                               70
P15C
Regulation of cell separation during the morphogenetic switch in Candida albicans.
Alberto González-Novo2, Pilar Gutierrez-Escribano1, David Caballero-Lima1, Carlos
R. Vázquez de Aldana2, Javier Jiménez2 and Jaime Correa-Bordes1
1
  Microbiology, Universidad de Extremadura, Avda Elvas s/n, Badajoz 06071, SPAIN,
Phone: +34 924289300 ext 6874, FAX: +34 924289300, e-mail: jcorrea@unex.es 2 Dpto.
Microbiología Genética, IMB. Universidad de Salamanca/CSIC.

In yeasts, cell separation requires localized degradation of the septum. This process depends on
the transcription factor Ace2, which activates the expression of several genes involved in cell
wall hydrolysis. In S. cerevisiae, the Mob2/Cbk1 kinase complex, a downstream effector of the
RAM signaling pathway, is required for the specific accumulation of Ace2 in daugther nuclei
at the end of mitosis. We have shown that Candida Cdc14 phosphatase is translocated to the
septum region of yeast-form cells at the end of mitosis, and is required for the activation and
daugther-specific accumulation of Ace2. Moreover, hypha-inducing signals abolished the
targeting of Cdc14 to the division plate. In this report, we suggest that the role of Cdc14 in
Ace2 activation is upstream of Mob2/Cbk1 activity, since in cdc14delta cells Mob2 is highly
modified and no Cbk1 kinase activity was detected. We have also investigated the role of
Candida septins in the regulation of cell separation, since this family of conserved proteins are
involved in the control of cytokinesis. We used FRAP analysis to characterize the dynamics of
the septin ring during yeast and hyphal growth. Our results indicated the existence of a septin
ring core, formed by the essential septins Cdc3 and Cdc12, with a very low exchange of
subunits with the cytoplasmatic pool (immobile state) that was independent of the type of
growth. However, the non essential Cdc10 septin ring changed from an immobile state to a
remarkably dynamic state during the yeast/hyphal switch in a Shs1/Sep7 dependent manner.
Cells from shs1delta mutant produced filaments in which the Cdc10 septin ring was blocked in
an immobile state that recruited the Cdc14 phosphatase to the septum. The lost of the dynamyc
Cdc10 ring turnover in shs1delta hyphae gave rise to the activation of cell separation in
subapical cells of the filament. Thus, these observations suggest that the septin Shs1/Sep7 is
required for modifying the Cdc10 septin ring stability and inhibition of cell separation during
hyphal growth in C. albicans.




                                                71
P16A
Roles of mRNA turnover factors in surface-attached growth in yeast
Ana Traven and Jorg Heierhorst
Molecular Genetics, St. Vincent's Institute, Princes Street, Fitzroy, Melbourne, Victoria 3065,
AUSTRALIA, Phone: +61 (0)3 9288 2480, FAX: +61 (0)3 9416 2676, e-
mail: atraven@svi.edu.au, Web: http://www.svi.edu.au/index.cfm?objectID=CA28A1CA-
A834-C4B3-CF1753DF70F1F896

Yeast cells can grow as differentiated multicellular structures when attached to a solid support.
Examples of this mode of growth are colonies growing on agar plates, as well as more
differentiated and complex biofilms that can form on different types of surfaces. Development of
these structures is accompanied by extensive changes in gene expression. Here we show that
regulation of mRNA turnover pathways might play a role in setting transcript levels during colony
and biofilm differentiation. We analysed mutants in the S. cerevisiae Ccr4-NOT mRNA
deadenylase that catalyses the first step in mRNA degradation and found that deletion of CCR4,
which encodes the deadenylase catalytic subunit, leads to cells that form colonies that are smaller
in size (even relative to slower growth rates of ccr4delta mutants in liquid cultures) and irregular
in shape, as opposed to bigger and mostly round-shaped wild type colonies. This phenotype is
mimicked by the Ccr4 catalytic domain mutant ccr4-1, demonstrating that the mRNA deadenylase
activity of Ccr4 is critical for this function. Mutants in some of the Not proteins (not1-1, not2-1
and not4delta) also give rise to colonies with similar morphology to those formed by ccr4
mutants, further proving a role for Ccr4-NOT during colony development. We also tested the role
of Ccr4 in biofilm formation by using the Sigma1278b strain background. Sigma1278 forms
differentiated mats when grown on semi-solid surfaces in a manner dependent on the Flo11
adhesin and is therefore used as the baker’s yeast model for fungal biofilm formation. ccr4delta
mutants formed mats smaller in size, which also appeared less structured than the wild type mats,
but more differentiated than flo11delta mats. These results suggest a possible role for Ccr4-NOT
during yeast biofilm formation. Notably, the Ccr4-NOT complex is conserved in Candida
albicans and it has recently been implicated in hyphal growth, biofilm formation and virulence.
Therefore, deciphering the roles of Ccr4-NOT during surface-attached growth in the well-
characterised Saccharomyces cerevisiae system will likely shed important insight into biofilm
formation in Candida albicans.




                                                72
P17B
Role of the Heat Shock Transcription Factor in the fungal pathogen, Candida albicans
Susan Nicholls and Alistair Brown
Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD,
Scotland, UK, Phone: 01224 555888, FAX: 01224 555844, e-mail: s.nicholls@abdn.ac.uk

The ability of pathogenic microbes such as Candida albicans to sense and respond rapidly to
changes in the growth environment is critical. C. albicans has evolved a variety of defences,
including specific stress responses, to evade destruction by the immune system and adapt to
changing microenvironments during disease establishment and progression. However, the
cellular mechanisms underlying such responses are poorly understood. It has become clear
that, whilst many signalling molecules and transcription factors are conserved in C. albicans,
the cellular roles of these regulators have diverged in comparison with the benign model yeasts
S. cerevisiae and S. pombe. All organisms respond to environmental stress by activating the
expression of proteins that protect them against the stress and repair any damage caused by the
stress. The classic “heat shock response” includes the induction of heat shock proteins, many
of which are chaperones involved in protein folding, trafficking and degradation. C. albicans is
obligatorily associated with its mammalian host, occupying niches where the temperatures are
generally maintained within homeostatically controlled limits. Yet a heat shock response has
been conserved during the evolution of this pathogen. Why? We predict that the heat shock
apparatus helps to protect the fungus against other stresses it encounters within the host and
therefore have targeted the Heat Shock Transcription Factor (HSF) for analysis. This factor
plays a central role in the regulation of the heat shock response in S. cerevisiae and is essential
for growth at normal temperatures. We have identified a single unnamed and unstudied locus
(orf19.4775) in the C. albicans genome sequence with strong sequence similarity to S.
cerevisiae HSF1. Our aim is to define the essential function(s) of Hsf1 during growth at normal
temperatures in addition to its predicted role(s) in C. albicans stress responses. We have
generated a doxycycline-conditional hsf1 mutant and shown that HSF1 is an essential gene in
C. albicans. Additionally, using HSE-LacZ reporter fusions we have demonstrated that heat
shock elements are functional in C. albicans, indicating that a functional Hsf1-HSE regulon
has been conserved in this pathogen.




                                                 73
P18C
Farnesol beyond morphogenesis control: effect in Non- Candida albicans Candida species
Margarida Martins, Mariana Henriques, Joana Azeredo and Rosário Oliveira
IBB-Institute for Biotechnology and Bioengineering, Universidade do Minho, Campus de
Gualtar, Braga 4710-057, Portugal, Phone: +351 253604400, FAX: +351 253678986, e-
mail: margarida.martins@deb.uminho.pt

Candididasis is one of the most important life-threatening opportunistic mycosis mainly occurring
in individuals with impaired immunity. Although Candida albicans remains the most common
fungal isolate, an increase in Non-Candida albicans Candida (NCAC) species is being reported.
In fact, Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis are
emerging as clinically relevant pathogens. So it is of great importance to study the mechanisms of
infection by these new species. Recently, farnesol, a quorum sensing molecule in Candida
albicans has been the focus of intense study concerning its effect in Candida’s virulence and
consequently its potential application as therapeutic agent. Nevertheless, to date, the action and
role of farnesol within Candida genus is yet not known. In this sense, the aim of this study is to
gain insights into the effect of farnesol in NCAC species. Accordingly, the effect of farnesol on
Candida glabrata, Candida krusei, Candida parapsilosis and Candida tropicalis reference strains
morphology and growth was evaluated. To assess morphological alterations, cells were grown
overnight in RPMI medium supplemented with 150 micromolar farnesol and inspected under
contrast light microscopy, after overnight growth. Candida species farnesol susceptibilities were
assayed at 0.5, 5, 50, 100 and 150 micromolar. Growth medium, farnesol solutions and inocula
were prepared following the recommendations outlined by the National Committee for Clinical
Laboratory Standards M-27A adapted to micro-dilution. The obtained results show that, at the
concentrations assayed, farnesol has an antifungal activity against NCAC species, with different
susceptibility profiles. Additionally, surviving cells exposed to the highest farnesol concentrations
did not present morphological alterations comparing to controls. These findings show that the
quorum sensing molecule, farnesol, has distinct species-specific effects, different from those
described for Candida albicans. Moreover, the results presented herein suggest that farnesol may
play a pivotal role in inter-species growth control, namely within mixed Candida species cultures
by the regulation of different cellular processes.




                                                 74
P19A
Characterisation of calcium signalling by gene knock-out and microarray analysis in
Aspergillus fumigatus
Omar Loss, Alexia Barberat, Herbert N Jr Arst and Elaine Bignell
Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London
SW7 2AZ, UK, Phone: 00 44 207 534 5293, FAX: 00 44 207 594 3076, e-
mail: o.loss04@imperial.ac.uk

In recent years Aspergillus fumigatus has become the most prevalent air-borne fungal
pathogen, causing severe and usually fatal invasive infections in immunocompromised hosts in
developed countries. Current understanding of calcium (Ca2+) homeostasis in A. fumigatus is
very poor although calcineurin, a highly conserved Ca2+-calmodulin-activated protein
phosphatase, has been shown to be involved in the control of the growth, morphology and
moreover the pathogenicity of A. fumigatus [Steinbach et al. ]. In addition some fungicidal
activities mediated by drugs and immunosuppressants are demonstrated to be mediated by
calcium, for example FK506, a specific inhibitor of calcineurin, renders the fungistatic
fluconazole fully lethal to fungal cells. In S. cerevisiae, the roles of transporters, including
pumps (Pmr1 and Pmc1), channels (Cch1, Mid1, and Yvc1), and exchangers (Vcx1) involved
in the calcium homeostasis and signalling are well understood. In addition the action of
calcineurin is well known. Supported by such findings in S. cerevisiae we are currently
investigating calcium homeostasis in A. fumigatus, facilitated by the publication of the whole
genome. Recent studies identified several calcium homeostatic genes whose transcription is
more than 1.5 fold up-expressed in vivo during murine invasive aspergillosis [Bignell et al.,
unpublished]. Based upon these data we have chosen to disrupt two specific stages of calcium
signalling, the entry of calcium into the cytoplasm, by disrupting each component of the
channel-forming complex encoded by cch1 and mid1, and the removal of calcium from the
cytoplasm into the vacuolar compartment, by knocking-out pmc1, encoding the vacuolar
calcineurin-activated calcium-transporting ATPase. The genes were deleted using a split
marker recombination system mediated by a loxP-phleo/tk marker module allowing dominant
selection of transformants due to resistance to phleomycin as well as dominant (counter)
selection of Cre recombinase-mediated marker excision event [Krappmann et al. ]. The
repertoire of A. fumigatus genes under calcium control was then assessed by microarray
analysis in collaboration with the European BIOCHIPS consortium (Toulouse, France) by
determining calcium exposure-mediated transcription in calcium-rich compared to calcium-
deplete conditions. 1. Steinbach, W, et al., (2006), Eukaryot. Cell, 5, 1091 2. Krappmann, S, et
al., (2005), Eukaryot. Cell, 4, 1298




                                                75
P20B
First characterization of the Cnh1 antiporter function in Candida species
Yannick Krauke, Olga Zimmermannova and Hana Sychrova
Dept. Membrane Transport, Institute of Physiology AS CR, v.v.i., Videnska 1083, Prague 142
20, Czech Republic, Phone: +420241062120, FAX: +420296442488, e-
mail: krauke@biomed.cas.cz

The pathogenicity of the human pathogen Candida albicans is influenced by many different
external and internal factors; the external and internal pH together with potassium homeostasis
plays an important role among them. To maintain optimum intracellular pH and potassium
content, yeast cells posses different transport systems in their plasma membrane. One of them is
the Na+/H+ antiporter which could be, besides detoxification of sodium cations, involved in the
regulation of intracellular pH, plasma membrane potential, cell volume and cell response to
osmotic shock. To characterize the role of plasma-membrane Na+/H+ antiporters in the physiology
and pathogenicity of different Candida species, two approaches have been employed. 1) The
heterologues expression of C. albicans antiporter, encoded by the CNH1 gene, in S. cerevisiae
mutant lacking its own sodium and potassium exporters, revealed the broad substrate specificity of
Cnh1p and its involvement in the regulation of intracellular pH (Kinclova, O., et al., (2001), FEBS
Lett., 504, 11). An in silico investigation showed that genes homologues to CNH1 exist in three
other human pathogenic species (C. dubliniensis, C. glabrata and C. parapsilosis) as well. Cnh1
proteins from these three Candida species have been also functionally expressed in S. cerevisiae.
Expression of the three antiporters increased the ability of S. cerevisiae cells to tolerate high
external concentrations of alkali-metal-cation salts and GFP-tagging helped to localize all three
antiporters in the plasma membrane. In these experiments, cells expressing the Cnh1 antiporter
from C. parapsilosis showed the highest tolerance to salts and cells expressing C. dubliniensis
antiporter the weakest one. By measuring the efflux of Na+ and K+ we could show that the
increase in salt tolerance reflects the transport capacity of the antiporters. The broad specificity
(ability to export potassium) of all tested Candida antiporters suggest their complex role in cell
physiology. 2) Construction of Candida mutants lacking the genes encoding Cnh1 antiporters
(using the SAT1 deletion cassette; Reuß, O., et al., (2004), Gene, 341, 119) will help to
characterize the Cnh1p’s role not only in cell osmotolerance but also in the Candida species
virulence and pathogenicity. This work was supported by MRTN-CT-2004-512481 and MSMT
LC531.




                                                76
P21C
Characterization of genes of the cell wall integrity pathway of the human fungal
pathogen Candida glabrata.
Tobias Schwarzmüller, Walter Glaser and Karl Kuchler
Medical Biochemistry, Medical University of Vienna, MFPL, Dr-Bohr Gasse 9/2, Vienna
1030, Austria, Phone: +43(0)1 4277 61818, FAX: +43(0)1 4277 9618, e-
mail: Tobias.Schwarzmueller@meduniwien.ac.at

The fungal cell wall is required for maintaining cell morphology, conferring mechanical strength
against osmotic changes and other adverse conditions, as well as communication with the
environment. The Saccharomyces cerevisiae protein kinase C (PKC) pathway is a typical MAPK
pathway, essential for the response to various stresses such as heat stress, exposure to alpha-
mating pheromone or cell wall damage as imposed by drugs such as Caspofungin. Cell wall
damage triggers a protective response, modulating expression of genes important for cell wall
assembly, maintenance, modification or remodeling. The human fungal pathogen Candida
glabrata is closely related to S.cerevisiae regarding its genome structure and organization.
Successful colonization of hosts requires the ability to sense environmental changes caused by
defense mechanisms of the host’s immune cells. Because the PKC pathway may play an essential
role in the host adaptation of C.glabrata, we initiated studies on its function. Homologous PKC
genes of C.glabrata were identified by genome-wide bioinformatics and multiple sequence
alignments. Using the dominant marker SAT1, a fusion PCR approach was optimized producing
deletion cassettes for all non-essential PKC genes in C.glabrata. Correct genomic integration of
cassettes was verified by PCR and Southern blotting. Deletion strains were analyzed for their
phenotypes on a variety of different physiological and adverse growth condition, including cell
wall stress excerted by caspofungin (CF), congo red (CR), calcofluor white (CW) and caffeine
(C). Spotting assays demonstrated that Cgslt2D, Cgbck1D and Cgmkk1D mutants were
hypersensitive to CF, while CR and CW did not affect growth. To test if the deletions cause
changes in cell wall composition, strains were treated with Zymolyase. A strain lacking CgSLT2 is
hypersensitive to Zymolyase when compared to wild type strain. Surprisingly, a fks1D strain
lacking the ß-1,3-glucan synthase Fks1 was resistant to Zymolyase. Notably, various stresses
triggered a rapid activation of the key kinase CgSlt2, as shown by immunoblotting using anti-
phospho-44/42 antibodies. Hence, the Cg PKC pathway shows a similar response to CF as in
S.cerevisiae. However, Cg is hyperresistant to CR, CW and C when compared to Sc, implying
differences in the sensing or signaling driving the PKC pathway in the fungal
pathogen. Supported by the Vienna Science & Technology Fund (HOPI-WWTF) and the FP6-
EURESFUN Project.




                                               77
P22A
The effects of glucose on Candida albicans
Alexandra Rodaki, Brice Enjalbert, Neil Gow, Frank Odds and Alistair Brown
Aberdeen Fungal Group, University of Aberdeen, Forresterhill, Aberdeen AB25 2ZD, United
Kingdom, Phone: +441224555888, FAX: +4412224555844, e-mail: a.rodaki@abdn.ac.uk,
Web: www.abdn.ac.uk

The human fungal pathogen Candida albicans (C. albicans) shows considerable flexibility in
adapting to environmental change. This is due partly to the ability of this fungus to assimilate
various nutrients in the different host microenvironments it inhabits. Furthermore, C. albicans
cells have evolved various defences, including specific stress responses, to evade the human
immune system. Previously, our lab examined the effects of glucose on the S. cerevisiae
transcriptome. This confirmed that glucose exerts pleiotropic effects on gene expression in S.
cerevisiae (1). Given the contrasting niches of C. albicans and S. cerevisiae, these fungi are likely
to experience different patterns of exposure to glucose. Moreover, there are known to be
differences in metabolic regulation between the two fungi. Therefore, in this study, a transcript
profiling approach was used to study the global responses of C. albicans to a range of glucose
concentrations. C. albicans cells were shown to exhibit subtly different transcriptional responses
to glucose compared to the analogous responses of S. cerevisiae. The responses of genes involved
in central carbon metabolism were similar in these two yeasts. However, the expression of
ribosomal protein genes was not induced by glucose in C. albicans. Also, specific C. albicans
genes involved in adaptation to environmental stresses were up-regulated in response to glucose.
These changes in the transcript profile were reflected in corresponding changes in C. albicans
physiology. The data indicate that glucose responses are linked to adaptational responses to ionic,
oxidative and certain antifungal stresses in C. albicans. This might contribute to the success of this
fungus as an opportunistic pathogen. (1) Yin, Z., Wilson, S., Hauser, N.C., Tournu, H., Hoheisel,
J.D. and Brown, A.J.P. (2003) Glucose triggers different global responses in yeast, depending on
the strength of the signal, and transiently stabilises ribosomal protein mRNAs. Molecular
Microbiology., 48, 713-724.




                                                 78
P23B
The role of the vacuole in the cell cycle progression of hyphal forms in Candida albicans
Veronica Veses, Andrea Richards and Neil Gow
Molecular and Cell Biology, School of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD,
UK, Phone: 00441224555878, FAX: 00441224555844, e-mail: v.veses@abdn.ac.uk,
Web: www.abdn.ac.uk

Candida albicans is able to grow in three forms- budding yeast, pseudohyphal and true
hyphal, which have distinct differences in their cell cycle. Mainly, yeast and pseudohyphae
cells of Candida albicans resemble cell cycle organization Saccharomyces cerevisiae, while
hyphae exhibit a linear growth rate, in contrast with the exponential kinetics showed by other
filamentous fungi. This linear kinetics is because the subapical cells remain in G1 for several
cell cycles, as a result of the asymmetric inheritance of vacuoles, since the apical cell primarily
receives cytoplasm, reentering in the cell cycle immediately after cytokinesis, but the subapical
cell receives larger vacuoles, and may not form a branch for several cell cycles (Barelle et al.,
2003). Our studies indicate that vacuole volume may play a direct role in cell cycle progression
by influencing cell size regulated events. A model has been suggested in which vacuole
volume influences the re-entry into the cell cycle leading to hyphal branching (Barelle et al.,
2006). We hypothesize that arrested cells (with large vacuoles) may be below a threshold cell
size to execute the size-dependent Start function at the G1/S cell cycle checkpoint. To test our
hypothesis a collection of conditional mutants in the vacuole inheritance process has been
assembled. Analysis of this mutant collection has lead to several observations, including the
role of the vacuole in the hyphae development, and how changes in the volume –but not in the
functionality- of this organelle lead to changes in the length of the subapical cells G1 arrest and
in its size. An implication of our model is that the organelle volume of eukaryotic cells cell
must be considered in relation to the threshold cell sizes that must be achieved to advance the
cell cycle. Therefore vacuolation, hyphal growth and cell cycle are linked both genetically and
physiologically. Funding from BBSRC is acknowledged (BB/D011434). References: Barelle
C.J., E.A. Bohula, S.J. Kron, D. Wessels, D.R. Soll, A. Schäfer, A.J.P. Brown, and
N.A.R Gow. 2003. Eukaryot. Cell 2:398-410. Barelle, C.J., M.L. Richard, C. Gaillardin,
N.A.R. Gow, and A.J.P Brown. 2006. Eukaryot. Cell 5 (2): 359-367.




                                                 79
P24C
Carbon dioxide regulated expression of the C. albicans carbonic anhydrase protein
Marianne Bolstad and Fritz A. Muhlschlegel
Biosciences, University of Kent, Giles Lane, Canterbury CT27NJ, United Kingdom,
Phone: +44 (0)1227 82 3735, FAX: +44 (0)1227 76 3912, e-mail: mb259@kent.ac.uk

Candida albicans is an opportunistic human fungal pathogen, capable of switching morphology
between spherical yeast, pseudohyphal and true hyphal growth forms. The morphological switch
can, among other signals, be induced by physiological concentrations of CO2/HCO3-, which
stimulate adenylyl cyclase encoded by CYR1. Carbonic anhydrase, encoded by NCE103, has, in
this research, been shown to play a key part in this signalling pathway. Immunofluorescence
revealed that Nce103p is a cytoplasmic and nuclear protein, while Cyr1p is predicted to be a
peripheral membrane protein. The focus of this research has been to investigate the physical and
functional interaction between Cyr1p and Nce103p. Yeast 2-hybrid assays revealed a weak
physical interaction between the two proteins. The functional analysis involved investigating the
expression of Nce103p in air and 5.5 % CO2. Western blot revealed that Nce103p is expressed in
air, but lower protein levels are observed in 5.5 % CO2. Nce103p is therefore not required in an
environment with excess CO2, as a rapid hydrolysis to HCO3- happens spontaneously. In an
adenylyl cyclase mutant, Nce103p is expressed both in air and 5.5 % CO2. These data indicate that
Nce103p is derepressed in elevated concentrations of CO2 in the mutant. The question of interest
is therefore whether the expression of carbonic anhydrase is regulated by transcriptional or post-
translational modifications and its association to adenylyl cyclase. Quantitative real time RT PCR
revealed that expression of NCE103 is 1.75 folds up-regulated in air.




                                               80
P25A
Histone modifications regulate morphogenesis in C. albicans
Denes Hnisz, Tobias Schwarzmueller, Walter Glaser and Karl Kuchler
Medical Biochemistry, Max F. Perutz Laboratories, Dr Bohr-gasse 9/2, Vienna 1030, Austria,
Phone: +431427761812, FAX: +43142779618, e-mail: denes.hnisz@meduniwien.ac.at

Candida albicans is an opportunistic human pathogen that can cause superficial and systemic
infections. The remarkable phenotypic plasticity that allows C. albicans to adapt to various host
niches is a major virulence attribute. C. albicans can exist as a unicellular yeast and is able to form
multicellular pseudo- and real hyphae. Furthermore, its diploid cells that are homozygous for the
mating-type like locus (MTL) can undergo an epigenetic phase-transition termed the white-opaque
switching. Whereas white cells have a round shape and are unable to mate, opaque cells have an
oval shape and are mating-competent. While the role of transcription factors in phase changes is
widely investigated, little is known about epigenetic mechanisms underlying switching. During
the white-opaque transition, phase-commitment is dependent on a single master locus WOR1, but
it includes changes in the expression levels of about 400 genes. Also, switching frequencies
between the two phases are modulated by globally acting histone deacetylases Rpd3 and Hda1.
The major goal of our studies was thus to identify further histone modifying enzymes regulating
white-opaque switching and analyze their regulatory mechanisms. Using genome-wide
bioinformatics, we identified all putative histone-modifying genes of C. albicans otherwise known
from the Saccharomyces cerevisiae genome. We created the respective homozygous deletion
mutants in C. albicans, in a MTL homozygous background. We assayed the influence of the
deletions on the white to opaque and on the opaque to white transitions. This way, we established
functional categories for switching modulators. Quantitative mating assays show that all the
mutants maintain wild-type mating efficiencies in the opaque phase, arguing that the modulators
effect phase transitions specifically rather than globally. We demonstrate with real-time PCR
analyses that the expression of the modulators is independent of the master switch locus WOR1.
Our results suggest a new model of the regulation of white-opaque switching that includes at least
another so far unidentified second master regulator that has a mutual transcriptional dependence
on WOR1 to determine the phase outcome and the transcriptional feed-backs are mediated by
histone-modifying cofactors. Supported by the Vienna Science & Technology Fund (HOPI-
WWTF-LS133), the FP6-EURESFUN Project and the Vienna Biocenter PhD Program.




                                                  81
P26B
Homeostatic mechanisms regulate the amino acid biosynthesis transcription factor Gcn4
in Candida albicans
Tsvia Gildor, Einav Deutscher, Avigail Atir-Lande and Daniel Kornitzer
Molecular Microbiology, Technion, Israel, Efron 2, Haifa IL 31096, Israel, Phone: (0)-972-4-
8295258, FAX: 972-4-8295254, e-mail: tsvia@tx.technion.ac.il

The fungal pathogen Candida albicans adapts to changes in external conditions by morphological
and physiological changes. These responses require tight and rapid regulatory systems. Gcn4 is a
master regulator of amino acid biosynthesis in fungi that, in addition, affects cellular
morphogenesis in C. albicans. The classical activation mechanism of Gcn4 involves translational
induction upon activation of the kinase Gcn2 by amino acid starvation. In addition, upon
phosphorylation by the CDK Pho85 in conjunction with the cyclin Pcl5, Gcn4 is rapidly degraded
via the ubiquitin system. Under starvation conditions, Pho85/Pcl5 activity is reduced, causing
reduced phosphorylation, and therefore stabilization, of Gcn4. In C. albicans, tight regulation of
CaGcn4 activity is achieved via a series of positive and homeostatic negative feedback loops: first,
CaGcn4 is a transcriptional activator of its own gene. Second, the cyclin CaPcl5, which is
essential for CaGcn4 degradation, is transcriptionally induced by CaGcn4. Finally, the activity of
the cyclin CaPcl5 is self-limiting due to phosphorylation by the CDK molecule that it activates.
This latter regulation involves a novel mechanism: the phosphorylated cyclin loses affinity for its
specific substrate CaGcn4, and is then rapidly cleared from the CDK by ubiquitin-mediated
degradation. Phenotypic analysis of the Capcl5-/- mutant indicates that CaPcl5 also modulates the
filamentous response of C. albicans in amino acid-rich media. We hypothesize that the multiplicity
of regulatory mechanisms of CaGcn4 may reflect the diversity in responses induced by Gcn4.




                                                82
P27C
Functional complementation of S. cerevisiae by the C. glabrata KRH1 and KRH2 genes
Katrijn De Brucker and Patrick Van Dijck
Department of Molecular Microbiology, VIB, KULeuven, Kasteelpark Arenberg 31, Leuven
3000, Belgium, Phone: +32 (0)16 32 1500, FAX: +32 (0)16 32 1979, e-
mail: katrijn.debrucker@bio.kuleuven.be, Web: http://bio.kuleuven.be/mcb/

Candida species are considered a major cause of opportunistic infections in humans. Although C.
albicans is the most common Candida species, C. glabrata already causes 20% of systemic
candidiasis and 30% of urinary tract infections. C. glabrata is resistant to fluconazole, a common
used antifungal, and has a higher mortality rate compared to C. albicans. The genome of C.
glabrata shows a high degree of homology with S. cerevisiae and is haploid. In contrary to C.
albicans, C. glabrata has a normal codon usage. Recently Peeters and colleagues (2006)
discovered a mechanism in yeast by which the G alfa protein Gpa2 activates PKA through two
kelch-repeat proteins, Krh1 and Krh2, bypassing adenylate cyclase stimulation. Hence, Gpa2
regulates PKA activity via two distinct pathways: through stimulation of adenylate cyclase and
through inhibition of the Krh proteins. We investigated if the C. glabrata homologues of ScKrh1
and ScKrh2 can complement the respective deletion mutants of S. cerevisiae. By measuring the
trehalose content of the respective deletions mutants, transformed with CgKrh1 and CgKrh2
cloned into pBEVY-vectors, after 12, 24 and 48 hours, we showed that ChKrh1 and ChKrh2 can
complement the function of ScKrh1 and ScKrh2. We will also investigate the expression of
STRE-controlled genes and the formation of pseudohyphae in these transformed deletion
mutants. C. glabrata KRH1 and KRH2 deletion strains will be made. The morphology, trehalose
mobilisation and the expression of STRE-controlled genes in these mutants will be investigated.




                                               83
P28A
Localization of oxidative stress-related proteins Glr1, Gnd1 and Zwf1 in the human
fungal pathogen Candida albicans
Karin Strijbis1, Wouter Visser2 and Ben Distel1
1
  Department of Medical Biochemistry, Amsterdam Medical Center, Meibergdreef 15,
Amsterdam 1105 AZ, The Netherlands, Phone: +31205665132, FAX: +31306915519, e-
mail: K.Strijbis@amc.uva.nl 2 Department of Genetic Metabolic Diseases, Amsterdam Medical
Center

The oxidative stress defense machinery of the opportunistic fungal pathogen Candida albicans
plays an important role in its survival in the human body because the fungus is challenged by
reactive oxygen and nitrogen species (ROS/RNS) following phagocytosis by macrophages and
neutrophils. Glutathione reductase (Glr1) is a key enzyme in the protection against oxidative
stress, whose activity is dependent on NADPH produced by the pentose phosphate pathway (PPP).
Although the PPP is presumed to be a cytosolic pathway in most organisms, the two
dehydrogenases of this pathway, glucose-6-phosphate dehydrogenase (Zwf1) and 6-
phosphogluconate dehydrogenase (Gnd1), and also Glr1 all have putative peroxisomal targeting
signals (PTS) in C. albicans: Zwf1 has a putative PTS1 and both Gnd1 and Glr1 have a putative
PTS2. To determine the subcellular localization of Glr1 and the PPP enzymes, we performed
subcellular fractionation and Nycodenz density gradient analysis on C. albicans cultures grown on
oleic acid, as fatty acid beta-oxidation will result in the production of ROS in peroxisomes.
Enzyme assays showed that Glr1 localizes to the cytosol and to mitochondria and that a small but
significant part is present in the peroxisomal fractions. Glr1 enzyme activity is almost completely
lost in a GLR1 knockout, suggesting that the single GLR1 gene encodes the cytosolic,
mitochondrial and peroxisomal proteins. The enzyme activity of Zwf1 and Gnd1 was detected in
both peroxisomes and cytosol indicating their dual localization in the cell. A C. albicans knockout
strain of the PTS2 receptor PEX7 lost peroxisomal Gnd1 activity, whereas the localization of the
PTS1 protein Zwf1 was unaffected. mRNA analysis showed that GND1 is a spliced gene with the
putative PTS2 in its intron. Overexpression of the spliced and unspliced mRNAs confirmed that
they encode the cytosolic and peroxisomal Gnd1, respectively. These results indicate that the C.
albicans genes GLR1, GND1 and ZWF1 all encode proteins with a (partial) peroxisomal
localization, which may reflect the specific requirements of this organelle in its defense against
oxidative stress.




                                                84
P29B
Developing tools for generating gene disruptions in Candida parapsilosis
Chen Ding and Geraldine Butler
School of Biomolecular & Biomedical Science, Conway Institute, University College Dublin,
Belfield, Dublin D 4, Ireland, Phone: +353 1 7166838, FAX: +353 1 2837211, e-
mail: chen.ding@ucd.ie

Candida parapsilosis is the second most common cause of candidiasis, yet there are few studies of
its biology and virulence. The generation of biofilms by C. parapsilosis is considered as a pivotal
factor in infection, and there has been several reports of disease outbreaks associated with an
environmental source. However, the lack of genetic tools impedes the molecular analysis of
biofilm formation. Here, we describe the development of a gene disruption method based on the
Candida albicans SAT1 flipper cassette (Reuss et al, 2004). The original cassette contains a
nourseothricin resistance marker, SAT1, whose expression is driven from the ACT1 promoter, and
a FLP recombinase expressed from the MAL2 promoter. The cassette is surrounded by recognition
sites for the FLP recombinase. We were enable to use cassette directly in C. parapsilosis. In this
study, we substituted the ACT1 gene promoter and MAL2 promoter of C. albicans with the
equivalent sequences from C. parapsilosis. This construct was then used in a two-step approach to
replace both alleles of the URA3 gene in the C. parapsilosis type strain CLIB214. The cassette
was removed once the disruption was generated, yielding an auxotrophic ura3 mutant that in
otherwise almost identical to the wild type. We have also disrupted the C. parapsilosis orthologue
of the BCR1 gene, which is known to control expression of cell wall genes and biofilm
development in C. albicans. The phenotype of the CpBCR1 disruption is currently being
analysed. Reuss, O, et al., (2004), Gene, 341, 119




                                                85
P30C
Putative G protein-coupled receptors GPRC and GPRD are involved in development and
morphogenesis in Aspergillus fumigatus
Alexander Gehrke 1, Thorsten Heinekamp 1 and Axel A. Brakhage 1,2
Molecular&Applied Microbiology, Leibniz Institute for Nat.Product Research&Inf. Biology,
Beutenbergstr 11A, Jena 07745, GERMANY, Phone: +493641656815, FAX: +493641656825,
e-mail: alexander.gehrke@hki-jena.de, Web: www.hki-jena.de
1
  Leibniz Institute for Natural Product Research and Infection Biology - Department of Molecular
and Applied Microbiology – Hans-Knöll-Institute, 2Friedrich-Schiller-University, Jena

The opportunistic human-pathogen Aspergillus fumigatus was subject to recent studies on cAMP
signal transduction with regard to morphogenesis and virulence. To date, one of the most
important questions is still unanswered: what are the external signals and the corresponding
proteins sensing those ligands or stimuli which enable the fungus to grow in a wide variety of
different ecological niches? In a first approach, two genes encoding putative G protein-coupled
receptors gprC and gprD, designated as carbon source-sensing receptors, were deleted in A.
fumigatus. The physiological characterisation of the mutants revealed altered growth on solid
media. However, various growth conditions, which included the use of different carbon- and
nitrogen-sources, did not restore the defect of the mutants. Virulence of the mutant strains, as
tested in a low-dose murine infection model, is attenuated. The function of the putative GPCRs
was further investigated by analysing fluorescent protein-fusions in vivo by confocal
microscopy. Recent data on the function of the receptors will be presented.




                                              86
P31A
Glutathione transferases in Candida albicans and response against external stresses
Ana Garcerá, Lidia Piedrafita, Celia Casas, Alicia Izquierdo, Lina Barreto and
Enrique Herrero
Department of Basic Medical Sciences, Faculty of Medicine, University of Lleida, Montserrat
Roig 2, Lleida 25008, Spain, Phone: +34973702409, FAX: +34973702426, e-
mail: ana.garcera@cmb.udl.es, Web: http://www.udl.es/

Glutathione transferases (GSTs) conjugate xenobiotics to reduced glutathione, followed by
elimination of the conjugates from the cell. GSTs are also important in protection against
oxidative stress. GSTs are divided into classes based on sequence, substrate specificity or
immunological properties. Omega class GSTs diverge from other GST classes because they have
low activity against standard GST substrates, whereas they are active as glutaredoxins. Thus,
omega GSTs act as thiol redox regulators. Only a few fungal GSTs have been studied to some
extent, especially in S. cerevisiae. This yeast has two proteins with standard GST activity (Gtt1
and Gtt2), although they cannot be ascribed to established classes based on their sequences. Gtt1 is
associated to the endoplasmic reticulum. In addition, S. cerevisiae has a peroxisomal omega GST
involved in sulfur amino acid metabolism (Gto1) and two cytosolic omega GSTs (Gto2 and Gto3).
In silico analysis of the C. albicans genome reveals the existence of two ORFs coding for Gtt
homologues (orf 19.698 for CaGtt1 and orf 19.6947 for CaGtt2) and one ORF coding for a
putative omega GST (orf 19.2613 for CaGto1). CaGtt2 and CaGto1 have been purified by us from
recombinant E. coli cells and they display enzyme activity patterns typical of standard and omega
GSTs respectively. Expression of the three genes has been studied under environmental stresses.
While CaGTT1 is not expressed at detectable levels in any of the conditions tested, basal
expression is detected for CaGTT2 and CaGTO1 in exponential cells. CaGTT2 expression is
significantly induced by oxidative, osmotic, heavy metal and alkaline pH stresses, and in
stationary phase cells. Except for heavy metals, the other stresses also cause induction of CaGTO1
expression. Induction patterns show a complex dependence on CaHog1 and CaSko1 regulators,
while they are independent of Cap1. Filamentation conditions do not affect expression of both
genes. GFP-tagged versions of CaGtt2 and CaGto1 have been constructed and their cellular
localisation has been studied in vivo, either in exponential cells, in stationary phase cells and in
cells under oxidative or osmotic stress. Fluorescence levels parallel mRNA levels in the tested
conditions. CaGto1 displays a homogeneous cytosolic localization, while GFP-CaGtt2 forms
aggregates at buds and specific cytosol locations, which is compatible with actin patches
colocalization. Phenotypes of the homozygous mutants will be presented.




                                                87
P32B
Molecular bases for Paracoccidioides brasiliensis multiple budding cellular division: a
role for the small Rho-like GTPase Cdc42p.
Agostinho J. Almeida, Cristina Cunha, Belém Sampaio-Marques, Jenny A. Carmona,
Cecília Leão, Paula Ludovico and Fernando Rodrigues
Life and Health Sciences Research Institute (ICVS), University of Minho, Campus de Gualtar,
Braga 4710-057, PORTUGAL, Phone: +351 253604844, FAX: +351 253604831, e-
mail: ajalmeida@ecsaude.uminho.pt

The thermally dimorphic fungus Paracoccidioides brasiliensis grows as mycelia at
environmental temperatures whereas at 37ºC it assumes yeast-like morphology in host tissues.
The most distinctive feature of P. brasiliensis yeast form is its typical multiple budding,
however the mechanisms that underlie this phenomena are still unknown. This work focuses on
the study of this particular cellular division process and the biological events that trigger it. We
have isolated and characterized P. brasiliensis homolog to Saccharomyces cerevisiae Rho-like
GTPase Cdc42p (Pbcdc42p), a protein previously implicated in the production of multiple
buds in this eukaryotic model organism. Transformation of S. cerevisiae CDC42/cdc42
heterozygous diploid with Pbcdc42 under the control of a galactose-inducible promoter
showed an altered gemulation and bud scar pattern when compared to the empty vector,
indicating a probable dominant effect of Pbcdc42p. Tetrad dissection analysis further
established the role of Pbcdc42p by confirming functional complementation of S. cerevisiae
cdc42 null mutant. Our results show that both S. cerevisiae CDC42/cdc42 and cdc42 null
mutant transformed with Pbcdc42 present abnormal cellular morphologies, namely elongated
cells and the presence of more than one bud per mother cell. Bud scar calcofluor staining has
also revealed random bud site selection contrasting with normal bipolar or axial bud site
selection. Altogether, our preliminary analysis demonstrates that Pbcdc42p has an important
function in cell division control, particularly the regulation of temporal and oriental
organization. Future studies will be conducted to further characterize Pbcdc42p’s role in P.
brasiliensis multiple budding, namely gene silencing and screening of up- or down-stream
effectors of the molecular cascade. Acknowledgments Almeida, A. J. was supported by a
fellowship from Fundação para a Ciência e Tecnologia (FCT), Portugal (contract
SFRH/BD/8655/2002). This work was supported by a research grant from FCT (Grant
Number: POCTI/ESP/45327/2002).




                                                  88
P33C
Molecular and biochemical analysis of quorum sensing and intermicrobial
communication in the fungal pathogen Candida albicans
Fritz A. Mühlschlegel1, James A. Chaloupka2, Adam J. Mannan1, Lonny R. Levin2 and
Jochen Buck2
1
  Department of Biosciences, University of Kent, Giles Lane, Canterbury CT27NJ, UK,
Phone: +44 1227 823988, FAX: +44 1227 763912, e-mail: f.a.muhlschlegel@kent.ac.uk 2
Department of Pharmacology, Joan and Sanford I. Weill Medical College of Cornell
University, New York, USA

Candida albicans is part of the normal skin and intestinal flora in humans. A major determinant
for virulence is the reversible morphological transition between colonizing yeast forms and
invasive pseudohyphal or hyphal growth forms. In healthy individuals, fungal quorum sensing
molecules and interspecies communication from bacteria occupying the same niche help to
maintain C. albicans in its nonvirulent state. We investigate how these signals block the
morphological conversion to invasive forms by directly modulating the C. albicans adenylyl
cyclase. Both farnesol, a quorum sensing molecule from C. albicans, and C12-homoserine lactone,
a bioactive analog of a Pseudomonas aeruginosa quorum sensing molecule, inhibit purified C.
albicans Cdc35 adenylyl cyclase and prevent the yeast-to-filamentous transition. In both cases,
morphological conversion can be rescued by addition of membrane permeable cAMP. Exogenous
cAMP also restores hyphal formation blocked by fluconazole, a member of the most widely used
class of antifungal drugs. These data suggest that azoles, which are known to elevate endogenous
production of farnesol, indirectly modulate fungal cyclase activity.
Klengel T, Liang WJ, Chaloubka J, Ruoff C, Schröppel K, Naglik JR, Eckert SE, Mogensen EG,
Haynes K, Tuite MF, Levin LR, Buck J, Mühlschlegel FA (2005). Fungal adenylyl cyclase
integrates CO2 sensing with cAMP signalling and virulence. Curr. Biol. 15: 2021-2026.
Hornby JM, Jensen EC, Lisec AD, Tasto JJ, Jahnke B, Shoemaker R, Dussault P, Nickerson K.W
(2001). Quorum sensing in the dimorphic fungus Candida albicans is mediated by farnesol. Appl.
Environ. Microbiol. 67: 2982–2992.
Hogan D, Vik A, Kolter RA (2004). Pseudomonas aeruginosa quorum-sensing molecule
influences Candida albicans morphology. Mol. Microbiol. 54: 1212-23.




                                              89
P34A
The protein kinase Tor1 links nutrient sensing and filamentous growth in Candida
albicans
Robert J Bastidas, Maria Cardenas-Corona and Joseph Heitman
Department of Molecular Genetics and Microbiology, Duke University, 322 CARL Bldg.,
Research Drive, Box 3546, Durham NC 27710, United States, Phone: +001-919 684-2809,
FAX: +001-919 684-5458, e-mail: rjb10@duke.edu,
Web: http://mgm.duke.edu/faculty/heitman/index.htm

The life cycle of Candida albicans consists of two phases, a yeast phase and a filamentous phase.
The transition between yeast and filamentous growth is controlled by a variety of signaling
networks. In eukaryotic cells, the protein kinase Tor (Target of rapamycin) functions in a signaling
pathway implicated in regulating cellular responses to nutrients that is conserved from yeast to
humans. In this work, we have begun to characterize the function of C.albicans Tor1. Using
rapamycin, a pharmacological inhibitor of Tor, we find that CaTor1 is necessary for the ability of
C.albicans cells to switch between yeast and filamentous states. First, exposure to rapamycin
under non-filamentation inducing conditions elicits differential expression of genes involved in
filamentation, as detected by microarray analysis. Second, inhibition of CaTor1 enhances
flocculation during growth on filamentation inducing conditions. Third, northern analysis revealed
that CaTor1 is required for the repression of hyphal specific gene induction. Finally, a screen of
transcription factor mutants identified mutations in the filamentous growth repressors CaNrg1 and
CaTup1 that result in rapamycin hypersensitivity. Our results support a model in which CaTor1
couples nutrient sensing signaling cascades and filamentous growth possibly by governing the
activity of CaTup1 and CaNrg1.




                                                90
P35B
Purification and germination of chlamydospores produced by Candida albicans and
Candida dubliniensis.
Francesco Citiulo, Gary Moran, David Coleman and Derek Sullivan
Dublin Dental School & Hospital, Trinity College Dublin, Lincoln Place, Dublin 2, Ireland,
Phone: +353 (0)1 612 7275, FAX: + 353 (0)1 612 7295, e-
mail: francesco.citiulo@dental.tcd.ie

Candida dubliniensis is the most closely related species to Candida albicans. The two species
exhibit many phenotypic similarities and are difficult to discriminate in clinical samples. While
investigating the comparative growth characteristics of these two species we noticed that when
C. dubliniensis was incubated in the dark at room temperature on YNB agar supplemented with
0.025% (w/v) glucose and 20 mg/ml methionine, pH 4.3, after 5 days high levels of
pseudohyphae and copious amounts of chlamydospores were observed. These high levels of
chlamydospores were also produced in a liquid medium with the same composition. In the case
of C. albicans, high levels of chlamydospores were produced when strains were incubated in
liquid Corn Meal medium supplemented with 1% Tween 80 and 0.025% (w/v) galactose in the
dark at room temperature for 5 days. Selective disruption of yeast cells, hyphae, pseudohyphae
and suspensor cells using zymolyase has allowed us, for the first time, to generate pure
suspensions of chlamydospores from these species for further analysis. When mature
chlamydospores (approx. 15 days old) were purified and incubated in low nutrient-containing
medium and subsequently stained with FUN-1, the chlamydospores appeared to be
metabolically inactive or dormant. However, the addition of 2% (w/v) glucose and 10% (v/v)
serum to the suspension of chlamydospores resulted in a gradual appearance of red
fluorescence, indicative of a resumption of metabolic activity. After 48 h in this medium, these
reactivated chlamydospores were observed to bud and produce pseudohyphae and hyphae.
When dormant chlamydospores were supplemented with 0.025% (w/v) glucose and 20 mg/ml
methionine the chlamydospores were observed to produce small spherical thick-walled cells
(approx. 2 microns in diameter). Staining of these cells with DAPI suggests that they are
nucleate and following the addition of a supply of glucose and serum they also appeared to be
able to germinate. The ability to produce pure suspensions of chlamydospores will allow us to
perform further experiments to investigate the role of these structures in candidal biology.




                                                91
P36C
The GPI anchored Pga26 is required for cell wall architecture and morphogenesis in
Candida albicans
Leslie Laforet, Ana Martinez, Inma Moreno, Luis Castillo, Rafael Sentandreu and
Eulogio Valentin
Department of Microbiology and Ecology, University of Valencia, Vicente andres estelles av.
n/n, Burjassot-Valencia 46100, Spain, Phone: 34 96 386 46 57, FAX: 34 96 386 46 57, e-
mail: Leslie.Laforet@uv.es, Web: http://centros.uv.es/web/departamentos/D275/ingles/

Pga26 is a putative GPI-anchored protein (1,2), also known as orf19.2475, CA2885 and IPF
7204, of unknown function. Previous studies performed by our group found that PGA26 is
induced in protoplast during cell wall regeneration (3), for this reason we decided to select it
for a deeper study. The deduced amino acid sequence showed that Pga26 contains an N-
terminal hydrophobic signal; it is rich in serine and threonine (28.3%) amino acids, has a
putative N-glycosylation signal and a potential GPI (glycosylphosphatidylinositol) attachment
signal. To investigate the possible role of PGA26, construction of null mutant by targeted gene
disruption and analysis of the resulting phenotype was carried out. Homozygous pga26/pga26
had an increased sensitivity to calcofluor white, Congo red and zymolyase, when compared
with the parental strain, indicating changes in the cell wall. The analysis of the cell wall
polymers composition showed that the null mutant has a bigger amount of ß1,6 glucan than the
parental strain. These results suggest that the lack of Pga26 leads to a defective cell wall. It is
interesting to point out that the kinetic of the germ tube formation was increased in the null
mutant. We analyzed the behaviour of the homozygous mutant to different stress conditions.
The pga26/pga26 strain was more resistant to osmotic stress (CaCl2) and heat shock (55ºC)
than the parental strain. We also studied the effect of different drugs, such as Tunicamicin,
Amphotericin B and Ketoconazol. The null mutant was more resistant to Tunicamicin and
more sensitive to Amphotericin B and Ketoconazol than the parental strain.

REFERENCES
1. De Groot, P.W.J., et al., (2003). Yeast 20: 781.
2. Garcerá, A., et al., (2003). Microbiology 149: 2137.
3. Castillo, L., et al., (2006). Fungal Genetics and Biology 43: 124.




                                                 92
P37A
Deletion of Candida albicans SFL1 promotes hyphal development in sensing nutrient
deprivation
Jiangye Chen, Yandong Li, Xuming Mao, Chang Su and Fang Cao
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, SIBS,
CAS, 320 Yue-yang Road, Shanghai 200031, China, Phone: 86-21-54921251, FAX: 86-21-
54921011, e-mail: jychen@sibs.ac.cn, Web: http://www.sibs.ac.cn/

Candida albicans is one of serious opportunistic fungal pathogens of humans, which can cause
various forms of candidiasis ranging from superficial mucosal infections to life-threatening
systemic diseases, especially in patients with an immunocompromised system. One of important
properties of C. albicans known to contribute to its pathogenicity is the ability of its reversible
morphological transition from yeast to hyphal form. Both positive and negative control of
filamentous growth is important for virulence because both hyperfilamentous tup1 and
nonfilamentous flo8 mutant strains of C. albicans are avirulent. Multiple positive signaling
pathways have been well-characterized, including Cph1-mediated mitogen-activated protein
kinase (MAPK) cascade, Efg1 and Flo8-mediated cyclic AMP dependent protein kinase A (PKA)
signaling pathway. The negative control is mainly mediated by Tup1 through Rfg1 and Nrg1.
Lacking any one of these three regulators leads to constitutive filamentous growth and
derepression of hypha-specific genes under non-filament-inducing conditions. We identified a
homolog of S. cerevisiae Sfl1, CaSfl1, from Candida albicans, which could suppress the
flocculent and hypherfilamentous phenotype of S. cerevisiae sfl1 mutants. The CaSfl1 contains a
heat shock factor (HSF) like domain that could bind to inverted repeats of nGAAn called heat
shock elements (HSEs) within the target promoters. Consistent with its putative binding activity to
specific DNA via HSF domain, the CaSfl1 is localized in the nuclear independent of growth
forms, suggesting that the predicted function of CaSfl1 in the transcriptional regulation. Deletion
of both copies of SFL1 enhanced filamentous growth in nutrient poor media. Overexpression of
SFL1 led to a significant reduction of filaments formation. Consistent with their ability in
morphological transition, deletion or overexpression of the SFL1 attenuates virulence of C.
albicans in a mouse model. At lower temperature (25 oC), the sfl1 mutant cells could be developed
into short hyphae in nutrient limit media including SCLD, SLAD and Spider. Adding of serum in
the media has an additional activating effect on the germination, indicating that serum is an
additional input to activate the hyphal development at low temperature, integrates signals from
nutrient–sensing pathways to generate the final response.




                                                93
P38B
Molecular Cloning and Characterization of Candida albicans CZF2 gene
Huafeng Wang, Guanghua Huang and Jiangye Chen
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, SIBS,
CAS, 320 Yue-yang Road, Shanghai 200031, China, Phone: 86-21-54921152, FAX: 86-21-
54921011, e-mail: wanghuafeng@sibs.ac.cn, Web: http://www.sibs.ac.cn/

Candida albicans, the most frequently isolated fungal pathogen, switches heritably and at high
frequency between at least seven general phenotypes. The ability of morphological switching is
closely related with its pathogenicity. The most intensive studied morphological transitions are the
yeast-hyphae transition and white-opaque switch. Filamentous growth has also been observed in
the most popular model Saccharomyces cerevisiae. To determine whether a similar regulatory
mechanism exists in C. albicans, a C. albicans genomic DNA library was introduced into a S.
cerevisiae haploid flo8 mutant. In this screening, we identified several genes including CZF2 by
suppressing the invasive growth defect of flo8 mutant. The CZF2 gene has an ORF of 1875 bp,
encoding a deduced protein of 624 amino acids. Czf2 has a N-terminal conserved Zn2Cys6 zinc
cluster motif. The region forms a binuclear zinc cluster, in which two Zn atoms are bound by six
Cys residues. Ectopic expression of the CZF2 in S. cerevisiae partially suppressed defects in
filamentous and invasive growth of the mutants in filamentation MAPK pathway and flo8/flo8
mutant. To study the functional role of Czf2 in C. albicans, we disrupted both copies of CZF2
alleles. The czf2 null mutant showed slight defect of filamentous formation both in liquid media
and solid media. Compared to the wild strains, the czf2 mutant formed fewer branches on solid
serum-induced condition. In a systemic mouse model, czf2/czf2 mutant cells showed reduced
virulence comparing with wild type strains. In homozygous MTLa strain, deletion of CZF2
increased white-to-opaque transition efficiency obviously. The czf2 mutant also showed increased
mating efficiency. In efg1 mutants, expression of CZF2 was obviously up-regulated. These results
suggested the CZF2 contributes to C. albicans morphogenesis.




                                                94
P39C
Interactions among protein mannosyltransferase-isoforms of Candida albicans
Inga Schmidt, Steve Misselwitz and Joachim Ernst
Institut für Mikrobiologie, Heinrich-Heine-Universität, Universitätsstrasse 1, Düsseldorf
40225, Germany, Phone: +49 (0)211 8114833, FAX: +49 (0)211 8115370, e-
mail: Inga.schmidt@uni-duesseldorf.de

In fungi, secretory proteins including wall proteins are often O-mannosylated by protein
mannosyltransferases (Pmt proteins). In C. albicans the PMT gene family encodes five isoforms,
designated Pmt1, Pmt2, Pmt4, Pmt5 and Pmt6. Pmt1 and Pmt5 are homologous members of the
Pmt1-subfamily, Pmt2 and Pmt6 of the Pmt2-subfamily, while Pmt4 is the single representative of
the Pmt4 subfamily. In yeast and mammalian cells pair wise interactions of Pmt isoforms have
been described, some of which have been associated with transferase activity towards specific
substrates. In yeast interactions of Pmt1- and Pmt2-like isoforms, as well as Pmt4 homodimers are
known to occur, while homologues of Pmt2 and Pmt4 isoforms appear to functionally interact in
human cells. We used the split-ubiquitin system to probe interactions among all Pmt isoforms of
C. albicans. For this purpose, yeast strains producing Pmt fusions to ubiquitin CUB- and NUB-
portions were constructed. Using Pmt-CUB and -NUB fusions homodimer formation was detected
among Pmt4 and Pmt6 isoforms. The homodimer formation of Pmt4 was prevented by deletion of
its potential catalytic domain, while a point mutation of a conserved arginine (R137) did not have
any effect on homodimer formation. These results differ from interaction in Saccharomyces
cerevisiae, in which homodimer formation does not require the catalytic domain, while the
conserved arginine is essential.




                                                95
P40A
Living on a hostile ground: key transcription factors mediate stress response of Candida
glabrata.
Andreas Roetzer1, Christa Gregori2, Karl Kuchler2 and Christoph Schüller1
1
  Dpt. of Biochemistry, MFPL - University of Vienna, Dr. Bohrgasse 9/5, Vienna 1030,
AUSTRIA, Phone: +43 (0)1 4277 52805, FAX: +43 (0)1 4277 9528, e-
mail: andreas.roetzer@univie.ac.at, Web: http://www.mfpl.ac.at/ 2 Dpt. of Med. Biochemistry,
Div. of Mol. Genetics, MFPL - Med. University of Vienna, Dr. Bohrgasse 9/2, Vienna 1030,
AUT

To sense environmental conditions of different microenvironments conserved signal transduction
pathways mediate changes of transcription patterns to adjust metabolism, defense and adaptive
responses. Environmental stress response was originally defined in the related baker´s yeast
Saccharomyces cerevisiae, while nothing is known about C. glabrata environmental stress
response. In S. cerevisiae the transcription factors Msn2 and Msn4 are involved in response to a
variety of stresses. Msn2 rapidly shuttles from the cytoplasm to the nucleus in stressed cells and
regulates activation of about 200 genes. Other fungi such as S. pombe and C. albicans use stress
activated protein kinases and do not seem to have Msn2-like stress mediating factors. We found
that the environmental stress response of C. glabrata is regulated by S. cerevisiae-like Msn2
directed stress response mechanism. As key feature of the Msn2 orthologues we identified a
conserved motif or subdomain (MSD) shared by Msn2 orthologues from certain
Saccharomycotina. To prove this, we generated a fusion of C. glabrata Msn2 (CgMsn2) and cyan
fluorescent protein (CFP) protein and expressed it in yeasts and in C. glabrata. CgMsn2-CFP
behaved very similar to the S. cerevisiae Msn2-GFP fusion protein. We observed rapid and stress
regulated changes of its intracellular localization. We suggest that Msn2-like factors are important
not only for baker´s yeast but also for the very different environments of pathogenic
yeasts. Among different detractions that C. glabrata has to overcome, oxidative stress caused by
reactive oxygen species produced by leukocytes are a serious threat for the survival inside of the
host. The S. cerevisiae Yap1 and Skn7 transcription factors collaborate and are central in the
activation of genes to mount transcriptional response to oxidative stress. Here we show that
CgYap1 and CgSkn7 are involved in a defense mechanism similar to S. cerevisiae, and activate
important enzymatic defense mechanisms such as the C. glabrata catalase gene (CgCta1). Our
analysis highlights important similarities and subtle differences between yeasts related, yet
adapted to very different environment such as baker´s yeast and Candida glabrata.




                                                96
P41B
Cryptococcus neoformans response to hypoxia
Zuzana Moranova1, Jiri Pavlicek1, Misako Ohkusu2, Vendula Husickova1, Radek Novotny1,
Susumu Kawamoto2 and Vladislav Raclavsky1
1
  Department of Microbiology, Palacky University, Hnevotinska 3, Olomouc 77515, Czech
Republic, Phone: 00420 585 639 505, FAX: 00420 585 632 417, e-
mail: zuzana.moranova@post.sk 2 Research Center for Pathogenic Fungi and Microbial
Toxicoses, Chiba University, Japan

Oxygen is a growth-limiting nutrient for the obligate aerobic pathogenic yeast Cryptococcus
neoformans. A unique hypoxia-response has been described in C. neoformans culture only
recently, represented by cell cycle arrest partly in unbudded G2 as well as G1. The aim of our
work was to characterise this stress response in detail in order to help elucidate its physiological
role. When oxygen concentration drops in culture under conditions of limited aeration,
cryptococcal cells slow growth and delay the onset of budding to G2 that is gradually prolonged,
resulting in unbudded G2-arrest in some strains. Our results indicate that oxygen can also become
limiting in a well-aerated culture temporarily, i.e. during the late log phase of growth. Sensing
such limitation can enable the yeast to slow proliferation down timely to restore equilibrium in
oxygen supply and consumption, exploit all the nutrients available and, eventually, arrest in
response to nutrient limitation in G1. However this system can fail under specific conditions.
When a two-phase cultivation under constant hypoxia with a pulse of fresh nutrients and
temporary re-oxygenation is performed in rich medium, it results in a more uniform unbudded G2-
arrest of cells ready to restart growth rapidly after re-oxygenation. We show that some of the large
unbudded G2 cells are repeatedly able to escape the cell cycle arrest under sever hypoxia. This
happens when cells arrested in unbudded G2 are subjected to prolonged oxygen limitation (7-14
days). Under such conditions, few of the large unbudded G2 cells are able to escape the cell cycle
arrest. Once a mother cell escapes the arrest, it is able to give rise to daughters repeatedly, but
these daughters are unable to separate from their mothers, which results in peculiar clusters of
small drop-like daughters arranged around the budding site of their mother. We believe that
cryptococcal sensitivity to oxygen limitation and its hypoxia response may play role in its
pathogenicity and should be studied further. Recently we are striving to identify components
necessary for cryptococcal hypoxia response on molecular level using Agrobacterium mediated
insertional mutagenesis. Czech Science Foundation (310/06/0645) and Ministry of Education,
Youth and Sports, Czech Republic (MSM6198959216) supported this work.




                                                97
P42C
Comparative Analysis of EDT1 in Candida dubliniensis and Candida albicans
Leanne O'Connor, Derek Sullivan, David Coleman and Gary Moran
Oral Microbiology, Dublin Dental School and Hospital, Trinity College, Lincoln Place, Dublin
D2, Ireland, Phone: +353 (0)1 612 7350, FAX: +353 (0)1 612 7295, e-
mail: oconnorl@dental.tcd.ie

Candida albicans, the major fungal pathogen in humans is closely related to Candida dubliniensis.
C. dubliniensis is less virulent than C.albicans, produces fewer hyphae and is less tolerant of
environmental stress than C. albicans. The C.albicans gene EDT1 is a regulator of filamentation
and this gene is also essential for virulence during infection of Reconstituted human epithelial
(RHE) (Zakikhany, K., et al.,2006). An EDT1 gene was identified in the C.dubliniensis genome
that was found to be similar in length and the synteny of the EDT1 locus in both species was
identical. However following the alignment of CaEdt1p and CdEdt1p it was found that the
proteins shared only 22% homology. In this study the C.dubliniensis EDT1 gene was disrupted
and phenotypic analysis of the mutant was carried out. Results obtained from growing
C.dubliniensis deltaedt1/edt1 (Spider, YNB, SLD, YPS, Lees) suggest that CdEDT1 is not an
important regulator of filamentation in C.dubliniensis when grown on solid media, in contrast to
previous studies where deltaCaEDT1 produced no hyphae on RHE tissues. Hyphal induction was
also examined in liquid medium containing 10% serum, and results showed that the C.dubliniensis
deltaedt1/edt1 mutant produced hyphae more efficiently in response to serum. At 30 °C and 37°C
both the C.dubliniensis and the wild-type grew well with no noticeable differences in doubling
times. However when grown at 42°C the mutant had a shorter doubling time than the wild-type
(2.9 hrs vs 3.4 hrs). In addition growth curves carried out under high concentrations of salt (1.6M)
revealed that like C.albicans, the C.dubliniensis deltaedt1/edt1 mutant could grow at these
concentrations whereas the wild-type C.dubliniensis strain failed to grow. These data suggest that
deletion of CdEDT1 increases the resistance of C.dubliniensis to environmental stress and
enhances filamentation in serum. Further analysis will be carried out to compare the effects on
virulence of deletion of EDT1 in both species during RHE tissues and macrophage infection in
vitro in order to determine if divergent functions of these genes accounts for the difference in
virulence of these two species.




                                                98
P43A
Effect of Streptococcus mutans on Candida albicans biofilms
Ephie Kraneveld1, Tatiana Pereira1, Piet de Groot2, Arie Jan van Winkelhoff3, Bob ten Cate1
and Wim Crielaard1
1
  Cariology Endontology Pedodontology, Academic Centre for Dentistry Amsterdam,
Louwesweg 1, Amsterdam 1066 EA, The Netherlands, Phone: +31 20 5188596, FAX: +31 20
6692881, e-mail: E.Kraneveld@acta.nl, Web: www.acta.nl 2 Swammerdam Institute Life
Sciences, University of Amsterdam 3 Oral Microbiology, Academic Centre for Dentistry
Amsterdam

During aging an increase in Candida albicans can be observed in the human oral biofilm. In this
biofilm there are numerous interactions of the fungus with oral bacteria. To understand this
complexity we evaluated the effect of Streptococcus mutans (the most cariogenic bacterium in
dental plaque) on in vitro Candida albicans biofilm development. Biofilms of C. albicans ATCC
90028 were formed in single and dual species biofilms on hydroxyapatite (the main mineral
constituent of teeth) discs in an artificial saliva medium supplemented with glucose (0.2%).
Biofilms were grown for 24h and analyzed every two hours for the number of viable cells (CFU),
cell morphology (light microscope) and biofilm structure (confocal laser microscopy). Our results
show a statistical difference on dual species biofilm development for CFU counts of both species
when compared to single species (p<0.05). Confocal laser microscopy and light microscopy also
showed notable differences between dual species (fungal-bacterial) biofilms and single-species
biofilms. These are apparent in biofilm structure as well as in cell morphology of the micro-
organisms and can be an important clue towards understanding Candida pathology in the oral
cavity.




                                               99
P44B
The role of CdNRG1 in filamentation and virulence in Candida dubliniensis
Gary Moran1, Donna MacCallum2, Frank Odds2, David Coleman1 and Derek Sullivan1
1
  Division of Oral Biosciences, Dublin Dental School and Hospital, Lincoln Place, Dublin 2,
Ireland, Phone: +353 1 6127245, FAX: +353 1 6127295, e-mail: gpmoran@dental.tcd.ie 2
Aberdeen Fungal Group, College of Life Sciences & Medicine, University of Aberdeen

We have used the macrophage-like cell line RAW264.7 to study virulence factor expression in
C. albicans and C. dubliniensis. These experiments revealed that C. dubliniensis failed to
produce hyphae following phagocytosis by these cells, could not escape from the macrophage,
and was severely inhibited by co-culture with these cells. Attempts to stimulate hyphal
formation in C. dubliniensis with cAMP or a hyperactive allele of C. albicans RAS1
(RAS1G13V) did not promote filamentation. Recently, it has been shown that the negative
repressor of filamentation NRG1, is differentially expressed in C. albicans and C. dubliniensis
under some conditions (Staib, P. et al., (2005) Mol Micro, 55, p637-652). Expression analysis
of CaNRG1 by real-time PCR revealed that the mRNA was rapidly down-regulated in C.
albicans upon stimulation by 10% fetal calf serum and following phagocytosis by
macrophages. However, in C. dubliniensis, down-regulation of CdNRG1 was slower in 10%
serum, and only transient following phagocytosis by macrophages. In order to determine if
CdNRG1 expression was responsible for the non-filamentatous phenotype of C. dubliniensis
following phagocytosis by macrophages, we created a homozygous null mutant using the
SAT1-flipper. The nrg1 deleted strain was constitutively pseudohyphal at 30oC and 37oC.
Gentle sonication was used to separate these chains of cells to yield an inoculum of yeast cells
for experiments. Following phagocytosis of this mutant by macrophages, yeast cells rapidly
transformed to form true hyphae in a similar manner to C. albicans. The level of proliferation
of the nrg1 mutant was significantly higher than wild-type C. dubliniensis and was similar to
that observed for a wild-type C. albicans isolate. The virulence if this mutant is was assessed
using the systemic mouse model. Although both the C. dubliniensis parental strain and the nrg1
mutant could infect mice when challenged with a high inoculum, both strains exhibited
similarly low levels of virulence in this model, relative to C. albicans SC5314. These data
indicate that differential expression of the CdNRG1 regulator may explain the altered virulence
of C. dubliniensis in some models, but does not account for overall lower virulence of this
species in the systemic mouse model.




                                               100
P45C
Functional characterization of C. albicans WASP domains
Andrea Walther1, Yvonne Schaub2, Nicole Borth2 and Jürgen Wendland1
1
  Yeast Biology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen 2500, Denmark,
Phone: +45 3327 5230, FAX: +45 3327 4708, e-mail: anwa@crc.dk 2 Department of
Microbiology, Friedrich-Schiller-University, Jena and Junior Research Group: Fungal
Pathogens, Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll
Institute, Jena

The C. albicans homolog of the human Wiskott-Aldrich Syndrome protein, Wal1, is required
for filamentous growth in the human pathogenic fungus. Functional analysis of WAL1 has
shown that other defects of a WAL1 deletion are defects in endocytosis and the occurrence of a
fragmented vacuoles. Specific functions of Wal1 were investigated by a set of serial deletions
including the N-terminal WH1-B domains, the internal proline rich regions and the C- terminal
VCA-domain. The VCA domain, whic in S. cerevisiae is required for activation of the Arp2/3
complex, is not required for filamentation in C. albicans but its deletion increases vacuolar
fragmentation. On the other hand, the proline rich regions as potential substrates for the
interaction with other proteins are required for filamentation. The N-terminal WH1-B domain
is another potential protein interaction and localization domain. We will present our results on
the characterization of mutant phenotypes of different deletion alleles as well as on two-hybrid
interactions of WASP with potential regulatory proteins.




                                               101
P46A
Mechanisms of polarised growth in Candida albicans: The exocyst and SNARE
complexes.
Laura Ann Jones
Molecular biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10
2TN, United Kingdom, Phone: +44 (0)114 2222748, FAX: +44 (0)114 2222748, e-
mail: mbp05laj@sheffield.ac.uk, Web: www.shef.ac.uk

Candida albicans is the most prevalent human fungal pathogen. It is a polymorphic organism that
can grow in three different morphologies of yeast, pseudohyphae and true hyphae, and it is
thought that this is important for its virulence. By examining the localisation of Mlc-YFP we have
shown that highly polarised growth in true hyphal germ tubes is driven by a structure analogous to
the Spitzenkörper present in filamentous fungi. This structure is thought to be an accumulation of
secretory vesicles that have been transported to the tip along actin cables by the motor protein
Myo2 complexed to its regulatory light chain, Mlc1. In addition to this, a complex called the
polarisome is also present that acts to anchor and nucleate the actin cables. A key distinction
between the Spitzenkörper and polarisome is that Mlc1-YFP localises to an apical spot which is
clearly an internal structure and which appears as a ball when rendered in three-dimensions; in
contrast the polarisome is a surface crescent. Both the crescent and spot are present in many
hyphal tips but only the crescent is visible in yeast and pseudohyphae. In order to investigate
further the mechanism of vesicle delivery and fusion in hyphal tips we have examined the
localisation of exocyst components, Exo70 and Sec6 and the t-SNARE, Sso2. The exocyst is a
multi-protein structure where vesicles dock prior to a complex forming between the v-SNARE,
Snc1, on the vesicle and the t-SNARE on the plasma membrane which enables the vesicle to
finally fuse to the plasma membrane. Sec6-YFP and Exo70-YFP localise to a polarisome shaped
structure at the tip of hyphal cells; while the t-SNARE CaSso2-YFP localises to a Spitzenkörper-
like structure at the tip of the growing germ tube. Thus unexpectedly, Sso1 appears to be present
in the Spitzenkörper, while Sec6 and Exo70 are polarisome components. The presence of Sso2 in
the Spitzenkörper may indicate that vesicle fusion is occurring in the Spitzenkörper rather than the
plasma membrane. Alternatively, Sso1 may be delivered to sites of polarised growth by secretory
vesicles that accumulate in the Spitzenkörper. Evidence in favour of the latter hypothesis is that
Sso1 localisation disappears upon treatment with cytochalasin A, which disrupts actin cables.




                                                102
P47B
Sec2p, the GEF for the Rab GTPase Sec4p localizes to the Spitzenkörper in the human
fungal pathogen C. albicans.
Rachel Lane and Prof Peter Sudbery
Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank,
Sheffield S10 2TN, UK, Phone: +44(0)II42222748, FAX: +44(0)1142222748, e-
mail: MBP04RFL@SHEFFIELD.AC.UK

A striking feature of the human fungal pathogen C. albicans that is thought to be important for
pathogenicity is its ability to grow in three distinct morphological forms: yeast, pseudohyphae,
and true hyphae. In yeast and pseudohyphae polarized growth is driven by a structure that
resembles the polarisome of S. cerevisiae; however, germ tube extension in hyphae is driven by a
Spitzenkörper similar to that of filamentous fungi (Crampin et al, 2005). In S. cerevisiae secretory
vesicles, which carry the raw materials for the biosynthesis of new cell wall and for membrane
expansion at sites of polarised growth, are transported along actin cables by the motor protein
Myo2 complexed to its regulatory light chain Mlc1. These vesicles dock with a multiprotein
structure called the exocyst before fusion with the plasma membrane. Exit of vesicles from the
Golgi, transport along actin cables, and docking with the exocyst require the Rab GTPase Sec4,
which is activated by its GEF Sec2. Sec2 may also mediate the association of Mlc1 and Myo2
required for Myo2 activation. We report here that C. albicans Sec2-YFP localizes to the
Spitzenkörper and this localization is dependant on actin cables, providing the first direct evidence
that the Spitzenkörper is an aggregation of secretory vesicles. CaSec2 co-localises with CaMlc1,
which has previously been shown to localize with the Spitzenkörper (Crampin et al., 2005);
furthermore, we have detected a physical interaction between CaMlc1 and CaSec2 in vivo. We
have mapped a 58 aa domain within the C-terminus of CaSec2 that is required for its correct
localization. Full length CaSec2 is phosphorylated; however, a C-terminal truncation that lacks the
localisation domain is not phosphorylated, suggesting that phosphorylation of this domain is
required for correct localisation. In an attempt to identify the kinase responsible for CaSec2
phosphorylation we have investigated the role of the conserved NDR kinase Cbk1, which is not
only essential for hyphal formation in C. albicans, but whose homologues have been shown to be
absolutely required for polarised growth in S. cerevisiae (Cbk1), S. pombe (Orb6) and N. crassa
(Cot1). We have found that Sec2-6His co-immunoprecipitaes with Cbk1-YFP, indicating a
physical interaction in vivo. These results raise the exciting possibility that the aggregation of
secretory vesicles in the Spitzenkörper may be stimulated by a kinase cascade that targets Sec2.




                                                103
P48C
Environmental control of white-opaque switching in Candida albicans
Oliver Reuß, Yang-Nim Park, Knut Ohlsen and Joachim Morschhäuser
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg
97070, Germany, Phone: +49 (0) 931 312127, FAX: +49 (0) 931 312578, e-
mail: oliver.reuss@mail.uni-wuerzburg.de

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can
switch from the normal yeast cell morphology (white) to an elongated cell type (opaque), which is
the mating-competent form of the fungus. The ability to switch reversibly between these two cell
types also contributes to the pathogenicity of C. albicans, as white and opaque cells are differently
adapted to specific host niches. White-opaque switching is believed to be a stochastic process that
occurs spontaneously at a frequency of about 10-3, although at 37°C opaque cells are unstable and
revert to the white phase. We found that switching from the white to the opaque phase can also be
induced by environmental conditions. Transient incubation of white cells under anaerobic
conditions programmed the cells to switch en masse to the opaque phase even at 37°C, suggesting
that regulatory factors that respond to aerobic/anaerobic conditions are involved in
environmentally controlled white-opaque switching. Expression of the transcription factor CZF1,
which regulates filamentous growth of heterozygous MTLa/alpha cells under embedded, hypoxic
conditions, from a tetracycline-inducible promoter in the MTLalpha strain WO-1 resulted in mass
switching of white cells to the opaque phase and deletion of CZF1 in strain WO-1 abolished
anaerobically induced white-opaque switching. Therefore, Czf1p seems to be part of a signalling
cascade that controls switching of white cells to the opaque phase in response to anaerobic
conditions. Intriguingly, passage of white cells of strain WO-1 through the mouse intestine, a host
niche in which the cells are likely to be exposed to anaerobic conditions, resulted in a strongly
increased frequency of switching to the opaque phase. These results demonstrate that white-
opaque switching is not only a spontaneous process but can also be induced by environmental
signals, suggesting that switching and mating of C. albicans may occur with high efficiency in
appropriate niches within its human host.




                                                104
P49A
Control of ammonium permease expression and filamentous growth by the GATA
transcription factors GLN3 and GAT1 in Candida albicans
Neelam Dabas and Joachim Morschhäuser
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg
97070, Germany, Phone: +49 931 312127, FAX: +49 931 312578, e-
mail: Neelam.Dabas@mail.uni-wuerzburg.de

In response to nitrogen starvation, the human fungal pathogen Candida albicans switches from
yeast to filamentous growth. This morphogenetic switch is controlled by the ammonium permease
Mep2p, whose expression is induced under limiting nitrogen conditions. In order to understand in
more detail how nitrogen starvation-induced filamentous growth is regulated in C. albicans, we
identified the cis-acting sequences in the MEP2 promoter that mediate its induction in response to
nitrogen limitation.We found that two putative binding sites for GATA transcription factors have a
central role in MEP2 expression, as deletion of the region containing these sites or mutation of the
GATAA sequences in the full-length MEP2 promoter strongly reduced MEP2 expression. To
investigate whether the GATA transcription factors GLN3 and GAT1 regulate MEP2 expression,
we constructed mutants of the C. albicans wild-type strain SC5314 lacking one or both of these
transcription factors. Expression of Mep2p was strongly reduced in gln3 and gat1 single mutants
and virtually abolished in gln3 gat1 double mutants. Deletion of GLN3 strongly inhibited
filamentous growth under limiting nitrogen conditions, whereas inactivation of GAT1 had no
effect on filamentation, indicating that Gln3p has additional targets that are required for
filamentation. However, the filamentation defect of gln3 mutants could be rescued by constitutive
expression of MEP2 from the ADH1 promoter. In contrast, filamentation became independent of
the presence of a functional MEP2 gene in the gat1 mutants, indicating that the loss of GAT1
function results in the activation of other pathways inducing filamentous growth. These results
demonstrate that the GATA transcription factors GLN3 and GAT1 control expression of the MEP2
ammonium permease and that GLN3 is also an important regulator of nitrogen starvation-induced
filamentous growth in C. albicans.




                                                105
P50B
Analysis of the Candida albicans CAMP65 knock-out mutants
Silvia Sandini1, Roberto La Valle1, Flavia De Bernardis1, Caterina Macrì2 and
Antonio Cassone1
1
  Infectious, Parasitic and Immuno-mediated Diseases, Istituto Superiore di Sanità, viale
Regina Elena 299, Rome 00161, Italy, Phone: +39 (0)6 4990 2369, FAX: +39 (0)6 4938 7112,
e-mail: sandini@iss.it, Web: http://www.iss.it 2 Food Safety and Veterinary Public Health
dept.

The mannoproteins are the fungal cell wall components that play a main role in host-parasite
relationship. In particular Camp65p, a mannoprotein of 65 Kilodalton, belonging to the beta-
glucanase family, is the main target of the immune response against the human opportunistic
fungus Candida albicans. To investigate the role of this protein in the morphogenesis and
virulence of the fungus, we have constructed, by ura-blaster protocol, two independent sets of
CAMP65 knock-out mutants (CAMP65/camp65 heterozygous, camp65/camp65 null and
camp65/camp65-CAMP65 revertant strains). The correct construction of the mutant strains
was verified by PCR, Southern, Northern, Western-blot and immunofluorescence. The null
mutants, although viable, are inable to form hyphae, in vitro and in vivo, and to adhere to
plastic. Furthemore, the null mutants are significantly less virulent than the parental strain in a
systemic murine infection model, as shown by increased survival, lower CFU counts in the
kidney and organ histology. Moreover, in an estrogen-dependent rat vaginal infection model,
the early clearance of the fungus is also significantly more accelerated in the null mutants than
in the wild-type strains. Hyphal formation, adherence and virulence are restored in revertant
strains. To determine the effects of deleting CAMP65 on the integrity of the cell wall, we
detected the sensitivity of the cell wall-perturbing agents and the activation of MAP Kinase
pathway. The camp65 null mutants are clearly hypersensitive to Congo red, either in liquid or
solid medium, and weakly sensitive to SDS, tunicamycin and caffeine, only in liquid medium.
Moreover, after treatment with Congo red, we detected in the null mutants an increase in the
phosphorylation of Mkc1p, Cek1p and Cek2p (p42-44 homologues), consistent with the
increased sensitivity to this substance. In conclusion, these results show that the CAMP65 gene
is important for hyphal differentiation and appears to be critical for the virulence of Candida
albicans.

Sandini, S., et al., 2007, Cell Microbiol, e-pub 9/1/2007;
Navarro-Garcia, F., et al., 2005, Microbiology, 151(Pt 8), 2737;
Bates, S., et al., 2005, J Biol Chem., 280(24), 23408.




                                                 106
P51C
Translation and Coping with Stress: eIF2a kinases and the Cross-Pathway Control
system of Aspergillus fumigatus
Thomas Hartmann1, Elaine M. Bignell2, Gerhard H. Braus1, Sven Krappmann1
1
  Institut fuer Mikrobiologie und Genetik, Georg August Universitaet Goettingen,
Grisebachstrasse 8, Goettingen 37077, Germany, Phone: 0049 551 39 3821, FAX: 0049-551-
393330, e-mail: hartmann.tom@gmail.com
2
  Imperial College London, UK

Aspergilli represent unique pathogens with Aspergillus fumigatus being the predominant
perpetrator. We are interested in nutritional requirements sustaining propagation and supporting
virulence. Fungal amino acid (aa) biosynthesis is regulated on various levels; besides pathway-
specific systems, one global regulatory network has evolved that acts on aa metabolism as a
whole. In its very components, this Cross-Pathway Control is made up by an eIF2 kinase sensing
aa deprivation and translating it into increased levels of the transcriptional activator protein CpcA,
which in turn elevates transcription for the majority of amino acid biosynthetic genes. To elucidate
the role of the CPC signal transduction cascade, the gene of the sensor kinase CpcC was cloned. In
contrast to cpcAdelta mutants, strains deleted for cpcC are not impaired in virulence, indicating
that the basal expression level of CpcA is necessary and sufficient to support pathogenesis.
Western blot analyses indicate that the cpcC-encoded kinase is not required exclusively to
phosphorylate the eIF2 subunit. Upon inspection of the A. fumigatus genome, the presence of a
related gene (ifkB, for initiation factor kinase) could be revealed. The ifkB gene was deleted in and
corresponding mutant strains are currently evaluated.




                                                 107
P52A
Cyclin-dependent kinase regulation by the C. albicans CDK inhibitor Sol1
Ayala Ophir, Avigail Atir-Lande and Daniel Kornitzer
Molecular Microbiology, Technion Faculty of Medecine, 2, Efron St., Haifa 31096, Israel,
Phone: +972 (0)4 8295258, FAX: +972 (0)4 8295254, e-mail: danielk@tx.technion.ac.il

Candida albicans, an important human pathogen, is able to switch between yeast and hyphal
morphologies in response to environmental conditions. This ability is thought to contribute to C.
albicans pathogenicity. Cell cycle-dependent cellular morphogenesis is regulated in part by
cyclin-dependent kinase (CDK) activity in many eukaryotes, particularly in budding yeasts. The
C. albicans genome contain a Cdk1 homologue, three homologues of the S. cerevisiae G1 cyclins,
and only two B-type cyclins homologues, (as opposed to 6 B-type cyclins in S. cerevisiae). In
addition, Sol1, a CDK inhibitor (CDKI) analogous to the main S. cerevisiae CDKI Sic1, was
recently identified. The C. albicans sol1-/- mutant displayed a moderate growth defect and a long
delay in re-entering the cell cycle from stationary phase. Although Sol1 was identified as a
functional homologue of S. cerevisiae Sic1, the overexpression phenotypes of the two proteins
differ: Sic1 causes cell cycle arrest at G1/S in both C. albicans and S. cerevisiae, whereas Sol1
does not. Conversely, Sol1 has a stronger effect on morphogenesis than Sic1, inducing elongated
buds that resemble hyphal germ tubes. These results support a role for Sol1 in C. albicans
morphogenesis, and suggest that Sic1 and Sol1 have distinct cyclin-CDK specificities. To
understand the differences in the activities of these CDKIs, we first confirmed that Sol1, like Sic1
is localized in the nucleus. Next, we assayed in vitro the activities of different cyclin-Cdk1
combinations on generic CDK substrates, in the presence of increasing concentrations of Sic1 and
Sol1. We tested the S. cerevisiae M-phase cyclin Clb2 and S-phase cyclin Clb5, the C. albicans
mitotic cyclins CaClb2 and CaClb4, and the C. albicans G1 cyclins CaCcn1 and CaCln3. Our
results suggest a different inhibition pattern of Sic1 vs. Sol1 on ScClb2 and ScClb5, explaining the
differential effect of these CDKIs in S. cerevisiae. In C. albicans, there were no significant
differences in the CDK-cyclin specificity of those two CDKI for the mitotic cyclins, or for the G1
cyclin CaCcn1, but we did record a stronger inhibition of CaCln3 by Sic1. This difference
between Sic1 and Sol1 activity might account for the differential cell-cycle effect of these two
CDKIs, and ultimately might explain the relaxed coordination between morphogenesis and S-
phase initiation in C. albicans as compared to S. cerevisiae.




                                                108
P52A
Cyclin-dependent kinase regulation by the C. albicans CDK inhibitor Sol1
Ayala Ophir, Avigail Atir-Lande and Daniel Kornitzer
Molecular Microbiology, Technion Faculty of Medecine, 2, Efron St., Haifa 31096, Israel,
Phone: +972 (0)4 8295258, FAX: +972 (0)4 8295254, e-mail: danielk@tx.technion.ac.il

Candida albicans, an important human pathogen, is able to switch between yeast and hyphal
morphologies in response to environmental conditions. This ability is thought to contribute to C.
albicans pathogenicity. Cell cycle-dependent cellular morphogenesis is regulated in part by
cyclin-dependent kinase (CDK) activity in many eukaryotes, particularly in budding yeasts. The
C. albicans genome contain a Cdk1 homologue, three homologues of the S. cerevisiae G1 cyclins,
and only two B-type cyclins homologues, (as opposed to 6 B-type cyclins in S. cerevisiae). In
addition, Sol1, a CDK inhibitor (CDKI) analogous to the main S. cerevisiae CDKI Sic1, was
recently identified. The C. albicans sol1-/- mutant displayed a moderate growth defect and a long
delay in re-entering the cell cycle from stationary phase. Although Sol1 was identified as a
functional homologue of S. cerevisiae Sic1, the overexpression phenotypes of the two proteins
differ: Sic1 causes cell cycle arrest at G1/S in both C. albicans and S. cerevisiae, whereas Sol1
does not. Conversely, Sol1 has a stronger effect on morphogenesis than Sic1, inducing elongated
buds that resemble hyphal germ tubes. These results support a role for Sol1 in C. albicans
morphogenesis, and suggest that Sic1 and Sol1 have distinct cyclin-CDK specificities. To
understand the differences in the activities of these CDKIs, we first confirmed that Sol1, like Sic1
is localized in the nucleus. Next, we assayed in vitro the activities of different cyclin-Cdk1
combinations on generic CDK substrates, in the presence of increasing concentrations of Sic1 and
Sol1. We tested the S. cerevisiae M-phase cyclin Clb2 and S-phase cyclin Clb5, the C. albicans
mitotic cyclins CaClb2 and CaClb4, and the C. albicans G1 cyclins CaCcn1 and CaCln3. Our
results suggest a different inhibition pattern of Sic1 vs. Sol1 on ScClb2 and ScClb5, explaining the
differential effect of these CDKIs in S. cerevisiae. In C. albicans, there were no significant
differences in the CDK-cyclin specificity of those two CDKI for the mitotic cyclins, or for the G1
cyclin CaCcn1, but we did record a stronger inhibition of CaCln3 by Sic1. This difference
between Sic1 and Sol1 activity might account for the differential cell-cycle effect of these two
CDKIs, and ultimately might explain the relaxed coordination between morphogenesis and S-
phase initiation in C. albicans as compared to S. cerevisiae.




                                                109
P53B
The high osmolarity glycerol (HOG) response in the human fungal pathogen Candida
glabrata strain ATCC2001 lacks a signaling branch operating in baker’s yeast
Christa Gregori1,      Christoph Schüller2,  Andreas Roetzer2,      Gustav Ammerer2       and
              1
Karl Kuchler
1
  Medical Biochemistry, Medical University of Vienna, Max F. Perutz Laboratories, Dr.
Bohrgasse 9/2, Vienna 1030, Austria, Phone: +43 (0)1 4277 61818, FAX: +43 (0)1 4277 9618,
e-mail: christa.gregori@meduniwien.ac.at 2 Biochemistry & Molecular Cell Biology,
University of Vienna, Max F. Perutz Laboratories, Dr. Bohrgasse 9/5, Vienna 1030, Austria

The HOG MAP kinase pathway is required for the adaptation to high osmolarity stress in the yeast
S. cerevisiae. We investigate the function of a similar pathway in the human opportunistic fungal
pathogen C. glabrata. Cgsho1 deletion strains display severe growth defects under hyperosmotic
conditions, a phenotype not observed for yeast sho1 delta mutants. However, this phenotype is
restricted to C. glabrata strains derived from the ATCC2001 wild type strain, whose genome
sequence has been determined. Deletion of CgSHO1 in other genetic backgrounds fails to cause
osmostress hypersensitivity, whereas cells lacking the downstream Pbs2 MAP kinase remain
osmosensitive. Notably, ATCC2001 Cgsho1 delta cells also display methylglyoxal
hypersensitivity when compared to other C. glabrata genetic backgrounds, implying the inactivity
of the Sln1-branch in ATCC2001. Genomic sequencing of CgSSK2 in different C. glabrata strains
revealed that the ATCC2001 genome harbors a truncated and mutated Cgssk2-1 allele, the only
orthologue of yeast SSK2/SSK22 genes. Thus, the osmophenotype of ATCC2001 is caused by a
point mutation in Cgssk2-1, which debilitates the second branch of the HOG pathway.
Unexpectedly, and in contrast to yeast, Cgsho1 delta mutants also displayed hypersensitivity to
weak organic acids such as sorbate and benzoate. Functional complementation experiments
unequivocally demonstrate that HOG signaling in yeast and C. glabrata share similar functions in
osmostress adaptation. Moreover, CgSho1 is also implicated in modulating weak organic acid
susceptibilities, suggesting that HOG signaling in C. glabrata mediates response to multiple stress
conditions.




                                               110
P54C
Investigation into the role of the putative transcription factors CdCTA21 and CdCTA22
in Candida dubliniensis.
Tim Yeomans, Gary Moran, David Coleman and Derek Sullivan
Division of Oral Biosciences, Dublin Dental School and Hospital, Lincoln Place, Dublin 2,
Ireland,          Phone: 00353(0)16127366,             FAX: 00353(0)16127297,          e-
mail: tim.yeomans@dental.tcd.ie

Following the comparative genomic hybridization of C. albicans and C. dubliniensis DNA to C.
albicans DNA microarrays (Moran et al, 2004) over 200 genes were identified as either absent
from or significantly divergent in C. dubliniensis. Prominent among these was the CTA2 gene
family of C. dubliniensis and C. albicans. There are at present eleven CTA2 genes identified in the
C. albicans genome (CaCTA), but we have identified two in the C. dubliniensis genome
(CdCTA21 and CdCTA22). CdCTA21 is most homologous at the nucleotide level to CaCTA21
and CaCTA24 (77%) and CdCTA22 is most homologous to CaCTA26 (54%). Their function in
both organisms is largely unknown, although a one-hybrid screen using Saccharomyces cerevisiae
(Kaiser et al, 1999) suggested that they are likely to encode transcription factors. Expression of
CaCTA21 has previously been shown to be under the regulation of the morphogenetic regulator
Efg1p. The purpose of this study is to identify the role of the CTA2 gene family in C. dubliniensis
as there are only two members of this family. Our first objective was the deletion of the C.
dubliniensis CTA2 genes. We have successfully deleted CdCTA21 and CdCTA22 in WÜ284 using
the SAT1 flipper cassette method (Reus et al, 2004). Initial tests have shown morphological
differences between the wild type and CdCTA21 mutant. No phenotype has yet been observed for
the CdCTA22 mutant. On Pal’s agar at 30ºC, C. dubliniensis normally grows as pseudohyphae and
produces high levels of chlamydospores, however the CdCTA21 mutant does not produce
chlamydospores and the pseudohyphae are restricted in size. Further morphological tests in serum
have shown that the CdCTA21 mutant forms hyphae at a considerably reduced rate, compared to
the wildtype. In contrast to these results, on certain media where C. dubliniensis characteristically
grows in the yeast form (Spider medium and YEPD) the CdCTA21 mutant grows primarily in the
pseudohyphal form. These preliminary results suggest that CdCTA21 is able to both inhibit and
promote morphological change and thus may play an important role in hyphal formation. Current
work involves a comprehensive phenotypic analysis of the CdCTA21 and CdCTA22 mutants and
completion of the CdCTA21/22 double mutant. It is hoped that these studies with C. dubliniensis
will also shed light on the role of the CTA family in C. albicans.

Moran, G. et al, (2004), Microbiology 150 (10), 3363
Kaiser, B. et al, (1999), Yeast, 15, 585.
Reuss, O. et al, (2004), Gene, 341, 119.




                                                111
P55A
dentification of an N-acetylglucosamine transporter that mediates hyphal induction in
Candida albicans
Javier Alvarez2 and James Konopka1
1
  Dept. Molecular Genetics and Microbiology, State University of New York, Room 130 Life
Sciences Bldg., Stony Brook NY 11794-5222, United States, Phone: +1 631 632-8715,
FAX: +1 631 632-9797, e-mail: jkonopka@ms.cc.sunysb.edu 2 Graduate Program in Genetics

In response to environmental stimuli, the fungal pathogen Candida albicans undergoes a
morphological transition from budding to hyphal growth that underlies its ability cause infection.
One known inducer of this morphological change is the sugar N-acetylglucosamine (GlcNAc).
GlcNAc also plays an important role in nutrient sensing and cellular regulation in a wide range of
organisms from bacteria to humans. Therefore, proteomic comparison of plasma membrane
proteins from buds and from GlcNAc-induced hyphae was carried out to identify proteins
involved in mediating the switch to hyphal growth. One of the hyphal proteins (Ngt1) displayed
similarity to the major facilitator superfamily and was specifically induced by GlcNAc, but not by
other sugars or by serum. Ngt1 functions as a GlcNAc transporter since a C. albicans ngt1D
mutant was defective in GlcNAc uptake and heterologous expression of NGT1 in S. cerevisiae
promoted GlcNAc uptake. Transport mediated by Ngt1 was specific for GlcNAc and could not be
competed with other sugars. An Ngt1-GFP fusion was also induced following macrophage
phagocytosis, suggesting a role for GlcNAc in signaling entry into phagolysosomes. The ngt1D
mutant failed to induce hyphae at low GlcNAc concentrations, but high concentrations of GlcNAc
could bypass the need for NGT1 to induce hyphae. This suggests that elevated intracellular levels
of GlcNAc induce hyphal formation. Ngt1 is also interesting in that it represents the first
eukaryotic GlcNAc transporter to be discovered. The presence of NGT1 homologs in the genome
sequences of a wide range of eukaryotes from yeast to mammals suggests that they may also
function in other cellular processes regulated by GlcNAc, including those that underlie diseases
such as cancer and diabetes.




                                               112
P56B
Analysis of the hypoxic response in Candida albicans.
Siobhan Mulhern and Geraldine Butler
School of Biomolecular and Biomedical Research, Conway Institute, University College
Dublin, Belfield, Dublin D4, Ireland, Phone: 00353-(0)1-7166838, FAX: 00353 (0)1-2837211,
e-mail: siobhan.mulhern@ucd.ie

The pathogenic yeast Candida albicans produces filaments in response to hypoxic (low
oxygen) conditions, but the underlying molecular mechanism is poorly understood. Deleting
the transcriptional regulator efg1 increases filamentation in response to hypoxia, whereas
deleting ace2 abolishes filamentation, even in very low oxygen environments. Much of our
understanding of the hypoxic response comes from studies in S. cerevisiae, where the levels of
haem and of sterols are used to sense oxygen. Control is exerted via several transcription
factors, including Hap1, which regulates the expresson of the repressor Rox1 in response to
haem, and Upc2 and Ecm22 which regulate expression of a number of genes in response to
both haem and sterol levels. The C. albicans orthologue of Rox1 (Rfg1) has been shown to
play no role in the hypoxic response. We have tested a series of C. albicans strains carrying
knockouts of transcription factors (provided by A. Mitchell and D. Sanglard), and some
additional candidate genes, for defects in the filamentation response to hypoxia. We show that
deleting a homologue of HAP1 has no effect on filamentation in hypoxia. However, deleting
CaUPC2 (the sole orthologue of both ScUpc2 and ScEcm22) completely abolishes
filamentation. CaUpc2 is required for expression of several genes in the ergosterol pathway,
whose expression is induced by hypoxia. The UPC2 mutant strain also has a reduced growth
rate in hypoxia, we are therefore currently investigating the hypothesis that Upc2 is a major
regulator of the hypoxic response in C. albicans. Included in the mutants that lacked
filamentation in hypoxia were transcription factors that are also required for filamentation in
other inducing conditions (such as CPH2). Our assay also successfully identified mutations
with increased filamentation in hypoxia. These included BCR1, a gene shown to be required
for biofilm development, and RLM1, an orthologue of an S. cerevisiae gene involved in
maintenance of cell wall integrity. In other assays, the bcr1 knockout fails to respond to a
potential quorum sensing agent (i.e. filamentation is not inhibited). Our results suggest that
there is a strong correlation between regulation of cell wall biosynthesis and response to
hypoxia.




                                               113
P57C
Role of cAMP phosphodiesterases in Gpr1/Gpa2-mediated signalling in yeast
Alessandro Fiori and Lubomira Stateva
Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK,
Phone: +44      (0)161     275      1580,    FAX: +44       (0)161    275    1505,    e-
mail: Alessandro.Fiori@manchester.ac.uk

The cAMP pathway regulates yeast-to-hypha morphogenesis in Candida albicans, as well as
pseudohyphal development in Saccharomyces cerevisiae. It operates via cAMP, the levels of
which are determined by tightly regulated processes of synthesis catalysed by the adenylate
cyclase, and hydrolysis catalysed by two phosphodiesterases: with high (Pde2) and low (Pde1)
affinity for cAMP. Our previous work in C. albicans demonstrated that Pde2 plays a major role in
cell wall synthesis, hyphal development and virulence (Jung, W.H. and Stateva (2003), Yeast 22,
285; Jung, W.H. et al. (2005), Microbiol 49, 2961; Wilson et al, (2007), Mol Microbiol, in press),
whilst Pde1 plays a minor role in morphogenesis on certain media. However the latter enzyme, a
class II phosphodiesterase, regulates cAMP signalling in response to glucose stimulation and
intracellular acidification. Furthermore, we have demonstrated through epistasis analysis that
PDE1 genetically interacts with GPA2 and GPR1, and suggest that although Gpr1, Gpa2, and
Pde1 all function in the cAMP pathway, a distinct Gpr1/Gpa2-mediated pathway must exist that
governs growth and morphogenesis in a cAMP-independent manner. We are currently
investigating the genetic interactions involving the cAMP phosphodiesterases encoded by PDE2
and PDE1 with GPA2 and GPR1 in both C. albicans and the model yeast S. cerevisiae. The results
of the comparative phenotypic analysis of a series of isogenic strains with an emphasis on
hyphal/pseudohyphal development will be presented.




                                               114
P58A
Thiol-specific antioxidant-like protein (Tsa1p), a protein between cytoplasm and cell wall
Martina Brachhold1, Xin Xiong1, Constantin Urban2 and Steffen Rupp1
1
  Molecular Biotechnology, Fraunhofer Institute, Nobelstrasse 12, Stuttgart 70569, Germany,
Phone: +49      (0)711     970      4145,    FAX: +49       (0)711      970     4200,    e-
mail: Martina.Brachhold@igb.fhg.de, Web: www.igb.fraunhofer.de 2 MPI for Infection
Biology, Cellular Microbiology, Schumanstrasse 21/22, Berlin, Germany

The cell wall is the first contact site between host and pathogen and is thus critical for colonization
and infection of the host. We have identified Tsa1p as part of the hyphal cell wall and within the
cytoplasm of C. albicans. Tsa1p is a member of the ubiquitous Tsa/AhpC protein family. In S.
cerevisiae Tsa1p has been shown to be responsible for several distinct functions, including
functions in oxidative stress, heat shock and genome stability. We could show that Tsa1p fulfils
similar functions in C. albicans as well. Deletion of TSA1 in C. albicans results in a higher
sensitivity towards cell wall perturbing agents and in a change in expression levels of genes
encoding for cell wall proteins in hyphae. This indicates that Tsa1p is required for the appropriate
biogenesis of the hyphal cell wall. Tsa1p does not contain a typical signal sequence for entry into
the secretory pathway therefore the mechanism by which Tsa1p is released to the cell surface is
unknown. It might be secreted via a non-conventional pathway. For human Tsa1p, strong
interaction with the plasma membrane was reported indicating specific mechanisms directing
Tsa1p isoforms to the membrane. In further studies we will investigate how Tsa1p is localized to
the cell surface by elucidating the molecular determinants in Tsa1p required for its differential
localization as well as the signalling pathways inducing the change in localization.




                                                 115
P59B
Gene expression profiling and phenotypic characterization of Candida albicans MAC1
Marija Dukalska, Martina Brachhold, Nicole Hauser and Steffen Rupp
Molecular Biotechnology, Fraunhofer Institute, Nobelstrasse 12, Stuttgart 70569, Germany,
Phone: +49     (0)711     970     4171,     FAX: +49       (0)711     970     4200,    e-
mail: Marija.Dukalska@igb.fhg.de, Web: www.igb.fraunhofer.de

Copper and iron are essential trace elements for most organisms. They act as cofactors for many
metabolic or detoxifying enzymes. However, when present in excess, both metal ions can also be
toxic by generating damaging oxygen radicals. Organisms have evolved specific mechanisms to
control metal ion concentrations to ensure optimal conditions and avoid toxicity. High-affinity
iron uptake is required for the virulence of the opportunistic pathogen C. albicans. However, the
uptake of iron is dependant on the uptake of copper. The transcription factor MAC1 has been
shown to regulate the uptake of copper in C. albicans and strongly affect the iron metabolism. We
have investigated the response of C. albicans to high and low levels of copper, in the presence and
absence of Mac1p using transcriptional profiling. We could confirm that Mac1p regulates CTR1,
the central copper transporter in C. albicans. Furthermore we could show that copper imbalance
results in strong deregulation of iron uptake, cell surface proteins, the ergosterol pathway and
amino acid biosynthesis. Consequently we find enhanced sensitivity of mac1 kockout stains
against H2O2, Calcofluor White, Congo red as well as Amphotericin B. Similar as observed for S.
cerevisiae, Mac1p seems to regulate only few genes directly, however the impact on iron
metabolism results in strong secondary effects.




                                               116
P60C
Production of E,E-farnesol as a quorum sensing- molecule by Candida spp.
Kai Weber1, Bettina Schulz1, Reinhard Sohr2, Markus Ruhnke1 and Michael Fleischhacker1
1
  Molekulare Infektionsdiagnostik, Charité Universitätsmedizin Berlin, Charitéplatz 1, Berlin
10117, Germany, Phone: +49 (0) 30 450 51 33 04, FAX: +49 (0) 30 450 51 39 64, e-
mail: weber_kai@web.de, Web: www.charite.de 2 Charité Universitätsmedizin Institut für
Pharmakologie und Toxikologie, Dorotheenstr. 94 10117 Berlin, Germany, Phone: +49 (0) 30
450 513304

The pathogenic fungi of the genus Candida grow as budding yeasts or in a filamentous form
(dimorphism). Many of the species, especially C. albicans build biofilms on various surfaces. The
filamentous growth is induced by various factors e.g. the addition of serum to the growth medium.
The conversion from a single cell growth into the filamentous form is regulated by several
mechanisms and quorum sensing plays an important role. Autoinducers like E,E-farnesol (FOH),
which are called quorum sensing-molecule(s) (QSM) regulate growth and metabolism. FOH
regulates hyphae specific genes by stimulating repressor cascades. So far the knowledge about
non-Candida albicans and quorum sensing is very limited and a better understanding of this
phenomenon might result in new therapeutical options. We established a HPLC analysis to
measure the amount of derivatised FOH from culture supernatants of several Candida spp. which
were incubated in RPMI 1640 medium with and without fetal calf serum. The extraction and
derivatisation with 9-anthroylnitrile of this QSM was performed as previously described by Saisho
et al with slight modifications. We found that C. albicans produces the largest amount of QSM i.e.
up to 13ng/ l FOH (58microM) when cultured in serum-free conditions. The addition of fetal calf
serum to the cultivation medium lead to a decreased E,E-farnesol production and the
strongest.effects were observed in C. albicans and C. dubliniensis. All other non-C. albicans
species produced 0,2 ± 0,1ng/ l FOH (1microM). C. albicans and C. dubliniensis cell grew
dominantly in a filamentous form when cultured in RPMI 1640 with and without serum, whereas
all other strains grew predominantly as budding yeasts. We conclude from our experiments, that
signal molecules like FOH play an important role in a microbiological community, including
several different Candida spp. The high FOH concentrations observed in C. albicans and C.
dubliniensis lead us to conclude that this quorum sensing-system is especially important in these
species. The decreased amount of the quorum sensing-molecule in the presence of serum indicates
that the production of FOH is down-regulated in filamentous growth forms. A correlation between
virulence aspects and production of FOH was not observed. This conclusion is based on the
observation that filamentous growth results in an increased virulence and a high FOH
concentration inhibits the hyphal growth.




                                               117
P61A
Phenotypic Characterization and Regulation of Morphogenesis in Candida dubliniensis
Elena Lindemann, Michael Berg, Steffen Rupp and Kai Sohn
MBT, Fraunhofer IGB, Nobelstr. 12, Stuttgart 70569, Germany, Phone: +49 711 970 4145,
FAX: +49 711 970 4200, e-mail: elena.lindemann@igb.fraunhofer.de

Candida albicans and Candida dubliniensis both are opportunistic fungal pathogens that are not
only closely related but also display the same phenotypic characteristics including certain
virulence-related traits like the ability to undergo the yeast-to-hyphal transition or to adhere to
human epithelia. However, in contrast to Candida albicans, Candida dubliniensis is less
frequently associated with systemic diseases and shows a reduced virulence in animal infection
models. Using a two-dimensional genome display approach, a technique which we use for
molecular fingerprinting of diverse Candida sp., we found a significant difference in the resulting
spot patterns. This indicates that although both Candida species are phylogenetically closely
related, they differ considerably in their genomic organisation. Moreover, comparative phenotypic
studies using cell wall-pertubing agents like Congo red and the fluorochrome dye Calcofluor
White reveal a much higher sensitivity of Candida dubliniensis towards these agents both on
plates and in liquid media then Candida albicans. Apparently, both species seem also to alter in
their cell wall composition. Furthermore, we were able to identify an open reading frame in the
genome sequence of C. dubliniensis with significant homology to EFG1 in Candida albicans.
Both open reading frames share 74% homology indicating that this open reading frame might
represent the functional homolog of EFG1 in Candida dubliniensis. We could delete both alleles
of this open reading frame using the SAT1-flipper approach originally described for Candida
albicans. Deletion mutants were viable, indicating that this gene seems not to be essential. There
is also evidence that the mutant strains shows morphological defects e. g. different cell
morphology or reduced germ tube formation, similar to the situation for Candida albicans EFG1,
indicating that this gene represents a central regulator for morphogenesis in Candida dubliniensis.




                                               118
P62B
Characterization of a PRY-Family in Candida albicans
Marc Roehm, Elena Lindemann, Mascha Kallnischkies, Dijana Trkulja, Constantin Urban,
Steffen Rupp and Kai Sohn
MBT, Fraunhofer IGB, Nobelstr. 12, Stuttgart 70569, Germany, Phone: +49 711 970 4171,
FAX: +49 711 970 4200, e-mail: mro@igb.fraunhofer.de

Pathogenesis-related (PR-) proteins are transcriptionally upregulated when plants are
challenged with pathogens and are assigned to at least 14 different families. The members of
the PR-1 family are characterized by a stable, conserved (PR) domain and show antifungal
activity. Three proteins in S. cerevisiae share homologies to the PR-1 family and are therefore
named PRY1-PRY3 (pathogenesis-related proteins in yeast). In C. albicans, the PRY-family
consists of 5 homologues to PR-1 proteins. All PRY-family members comprise an N-terminal
hydrophobic signal sequence and the PR domain. The molecular function of these proteins is
yet not known. However, one of the PRY-family members in C. albicans, RBT4, has been
described as a virulence factor in a rabbit cornea model as well as in a systemic mouse model.
On the transcriptional level, RBT4 is repressed by TUP1 and upregulated in an EFG1-
dependent manner under hyphae-inducing conditions. In contrast, we found RBE1, another
member of the PRY-family, to be strongly de-repressed in efg1 mutants under various
conditions, including filamentous growth, indicating that EFG1 functions as a repressor for this
gene. In order to investigate on a molecular level the regulatory function of EFG1 for RBE1,
we analysed the promoter region of RBE1 for upstream regulatory sequences. We found an
approximately 1 kb region upstream of RBE1 sufficient for the transcriptional regulation
observed under physiological conditions. Within this sequence, we were able to define two
domains of ~250 bps in length, RI and RIV, with repressing and activating functions,
respectively, using lacZ-assays. Strikingly, the repressing domain RI is devoit of any putative
EFG1-binding site (E-box), suggesting that either EFG1 also binds to alternative consensus
sites or EFG1-mediated repression requires additional factors. In electrophoretic mobility shift
assays using crude cell extracts, both fragments show specific shifts, indicating that they
represent bona fide consensus sites for DNA-binding proteins. To further investigate the
molecular functions of Rbe1p and Rbt4p, we deleted the respective genes in the clinical isolate
SC5314. In preliminary virulence studies using a systemic mouse model, we found a moderate,
but yet significant reduction in pathogenesis in a rbe1/rbt4 double knockout strain.




                                               119
P63C
Regulation of Rac1 in Candida albicans
Hannah Hope, Robert Arkowitz and Martine Bassilana
, Institute of Signalisation Developmental Biology and Cancer, Parc Valrose, Nice 06108,
France, Phone: 0033 (0)4 92 07 6465, FAX: 0033 (0)4 92 07 6466, e-mail: hope@unice.fr,
Web: http://www.unice.fr/isdbc/

In the human opportunistic pathogen Candida albicans, morphological plasticity is critical for the
invasion and infection of host tissue. Rho G-proteins are essential regulators of cytoskeleton
organisation and cellular morphology in eukaryotic cells. In C. albicans it has been shown that
individual Rho G proteins control filamentous growth in response to specific stimuli. Cdc42 is
essential for filamentous growth in response to serum, while Rac1 is essential for filamentous
growth in response to an agar matrix environment (Bassilana, M. et al (2006) Eukaryot Cell
(5):321). We have demonstrated previously that Cdc24 is the specific activator for Cdc42 however
the Rac1 activator is unknown. We have identified and characterised two potential Rac1
activators. A BLAST search revealed two putative guanine nucleotide exchange factors (GEF) in
the C. albicans genome with similarity to the Ced-5, Dock180 and Myoblast city (CDM) family
which we have called DCK1 and DCK2. Members of this family all contain a DOCKER domain,
which is essential for exchange factor activity. DCK1 and DCK2 are expressed genes that are
adjacent to one another in the genome, and both contain a carboxy-terminal DOCKER domain.
Invalidation of DCK2 had no affect on budding growth nor filamentous growth in response to
serum or a matrix environment. In contrast, cells invalidated for DCK1 are unaffected for budding
growth yet are unable to filament in response to an agar matrix, similar to the phenotype of rac1
mutant cells . We verified this phenotype by deletion of the DCK1 and observed a similar
phenotype, defective in filamentation in an agar matrix ,yet filamentous in response to serum,
suggesting that this potential activator is specific for Rac1 and not Cdc42. This filamentous
growth defect of dck1 delta /dck1 delta cells can be partly overcome by overexpression of an
activated form of Rac1, Rac1[G12V], but not a dominant negative or wild-type form suggesting
that Dck1 is a Rac1 activator. Taken together, our results indicate that Dck1 and not Dck2 is a
GEF for Rac1 which is specific for filamentous growth. We are currently characterising this
exchange factor.




                                               120
P64A
YAK1 a Ser/Thr kinase involved in hyphal differentiation and maintenance in Candida
albicans.
Claire Naulleau1,      Sophie Goyard1,       Hélène Munier2,     Guilhem Janbon3       and
                    1
Christophe d'Enfert
1
  Fungal Biology and Pathogenicity, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015,
FRANCE, Phone: +33 (0)1 4061 3126, FAX: +33 (0)1 4568 8938, e-
mail: naulleau@pasteur.fr 2 Chimie Organique 3 Mycologie Moléculaire

Candida species, in particular Candida albicans and Candida glabrata, can form biofilms on
plastic surfaces. The inactivation of C. glabrata YAK1, which encodes a putative Ser/Thr kinase,
has a dramatic impact on biofilm formation. In Saccharomyces cerevisiae, the Yak1 kinase is
involved in glucose sensing. Here, we present the characterization of the C. albicans YAK1 gene
that encodes a homologue of the C. glabrata and S.cerevisiae YAK1. A C. albicans strain carrying
null mutations in the two CaYAK alleles was constructed. The mutant strain exhibited a strong
defect during the yeast to hypha transition under Efg1-dependent and -independent inducing
conditions. Consequently, biofilm formation by the C. albicans yak1 strain was dramatically
altered (10% of the biomass formed by a wild-type strain after 40 h of biofilm growth).
Quantitative real-time PCR was used to measure induction of hypha-specific genes (HSGs) in the
C. albicans wild type and yak1 strains. Results indicated that CaYak1 is necessary for HSG
expression. Serum is the most potent inducer of hyphae in C. albicans. In presence of serum in
liquid medium after 2 h of differentiation, the filamentation of the yak1 mutant was
indistinguishable of the wild type strain. Nevertheless, after a longer incubation time, 6 h, the wild
type strain continued to elongate, while the yak1 mutant exhibited abnormal patterns of
filamentation, including lateral and apical budding. This result, along with additional data obtained
using an ATP-analog sensitive allele of YAK1 indicates that Yak1 is involved in hyphal
maintenance. Attempts to link Yak1 to the cAMP–PKA pathway, a main signalling pathway for
the yeast-to-hypha transition, were unsuccessful. Indeed, addition of cAMP or over expression of
Tpk1, Tpk2 or Efg1, had no effect on the yak1 phenotype. To gain further insights in the Yak1-
dependent signalling pathway, a comparative phosphoproteomic analysis between the C. albicans
wild-type and yak1 strains was performed. Two proteins showed major changes in
phosphorylation but only minor or no change in protein abundance. These proteins were identified
as the 14-3-3 protein, Bmh1, and the putative thiol-dependent peroxidase, Tsa1. These two
proteins have been involved in the regulation of the yeast-to-hypha switch. Further evidences
linking these two proteins to CaYak1 will be presented.




                                                 121
P65B
In Candida albicans, maintenance of lipid rafts and the Spitzenkörper are
interdependent.
Peter Sudbery and Son Vi
Molecular Biology and Biotechnology, Sheffield University, Western Bank, Sheffield S10
2TN, United Kingdom, Phone: +44 (0)114 2226186, FAX: +44 (0)114 2222800, e-
mail: P.Sudbery@shef.ac.uk

A striking feature of the human fungal pathogen C. albicans, that is thought to be important for its
pathogenicity, is its ability to grow in three distinct morphological forms: yeast, pseudohyphae,
and true hyphae. Polarised growth in hyphae is driven by a Spitzenkörper, an accumulation of
secretory vesicles that forms a vesicle supply centre to focus the supply of secretory vesicles to the
growing tip. The Spitzenkörper can be conveniently visualised using high resolution fluorescence
microscopy by the localisation of Mlc1-YFP to a bright spot at or just behind the hyphal tip.
Polarised growth of the hypha also depends on an apical patch of lipid rafts. Lipid rafts are
membrane microdomains rich in sterols and sphingolipids that have been implicated in the
polarised growth in a wide variety of eukaryote cells. They can be conveniently visualised by
Filipin, a highly fluorescent polyene macrolide, which binds to sterols in lipid rafts. In C. albicans
it has been shown that the apical crescent of lipid rafts is disrupted by myriocin, an inhibitor of
sphingolipid biosynthesis; as a result hyphal growth is slowed and hyphal tips become swollen.
We have investigated the relationship between lipid rafts and the Spitzenkörper. Cells expressing
Mlc1-YFP were induced to form hyphae and then treated with myriocin. At intervals the hyphae
were briefly stained with filipin to monitor lipid raft polarisation and the integrity of the
Spitzenkörper monitored by the localisation of Mlc1-YFP. Loss of lipid raft polarisation
correlated with dissipation of the Spitzenkörper and cessation of polarised growth. Prolonged
filipin treatment and sub-lethal doses of the polyene amphotericin B also led to loss of polarised
staining which was also correlated with dissipation of the Spitzenkörper. We have previously
shown that Spitzenkörper maintenance is dependent on actin cables, which can be disrupted with
cytochalasin A. Treatment of growing hyphae with cytochalasin A resulted in a loss of lipid raft
polarisation. Thus the Spitzenkörper and polarisation lipid rafts are dependent on each other. We
propose a model in which lipid rafts directly or indirectly organise the formation and maintenance
of the Spitzenkörper; in turn the membranes of secretory vesicles, delivered to the hyphal tip via
the Spitzenkörper, are rich in lipid rafts.




                                                 122
P66C
Differential expression and activities of drug efflux proteins Cdr1p and Cdr2p in azole
resistant Candida albicans isolates
Ann Holmes, Ya-Hsun Lin, Kyoko Niimi, Erwin Lamping, Brian Monk, Mikhail Kenya and
Richard Cannon
Oral Sciences, University of Otago, 310 Great King St, Dunedin 9054, New Zealand,
Phone: +64 (0)3 479 7435, FAX: +64 (0)3 479 7078, e-mail: ann.holmes@otago.ac.nz

Fluconazole has become the antifungal drug of choice for non-life-threatening Candida infections.
The increasing clinical problem of drug resistance, however, most frequently results from
expression of drug efflux pumps Cdr1p and Cdr2p in the yeast cell membrane, although the
relative contribution of each pump is unknown. The aim of this study was to compare the relative
expression and function of the major C. albicans drug efflux pumps Cdr1p and Cdr2p in drug-
resistant C. albicans clinical isolates. Expression of Cdr1p and Cdr2p was determined, using
antibodies specific to each pump, by Western Blot analysis of purified plasma membrane
preparations from clinical isolates of azole-resistant C. albicans strains and from Saccharomyces
cerevisiae recombinant strains hyper-expressing either Cdr1p or Cdr2p. Immunoreactive proteins
were quantified by image analysis. Pump function was assayed by measuring glucose-dependent
efflux of the fluorescent pump substrate rhodamine 6G (R6G) from suspensions of glucose-
deprived yeast cells in the presence of RC21, a specific inhibitor of Cdr1p activity. Anti-Cdr1p or
anti-Cdr2p antibody reactivities on Western Blots were proportional to the amount of Cdr1p or
Cdr2p present in plasma membrane preparations. Cdr1p was found to be present in membranes of
two azole-resistant C. albicans strains at 8-10 times greater concentrations than Cdr2p. Peptide
RC21, (>0.612 M), completely inhibited R6G efflux from an S. cerevisiae strain hyper-
expressing Cdr1p but not from a Cdr2p-expressing strain. Glucose-dependent efflux of R6G from
a FLC-resistant C. albicans clinical isolate was inhibited by up to 66 per cent in the presence of
>0.3 M RC21. Conclusion: Both Cdr1p and Cdr2p are expressed in azole-resistant C. albicans
strains but Cdr1p is present in greater concentrations and contributes the most pump activity. This
research was supported by the National Institutes of Health, USA (R21DE015075-RDC;
R01DE016885-01-RDC). Y-H.L. was supported by an Otago Medical Research Foundation
Summer Studentship.




                                               123
P67A
Structure and function analysis of CaMdr1p, a MFS antifungal efflux transporter protein
of Candida albicans: identification of amino acid residues critical for drug/H+ transport.
Ritu Pasrija and Rajendra Prasad
School of Life Sciences, Jawaharlal Nehru University, New Delhi, New Delhi 110067, INDIA,
Phone: +91-11-2670-4509, FAX: +91-11-2671-7081, e-mail: ritupasrija@yahoo.com

We have cloned and overexpressed multidrug transporter CaMdr1p as a GFP tagged protein to
show its capability to extrude drug substrates. The drug extrusion was sensitive to pH, energy
inhibitors, and displayed selective substrate specificity. CaMdr1p has a unique and conserved
‘antiporter motif,’ also called ‘motif C’, [G(X)6G(X)3GP(X)2GP(X)2G] in its transmembrane
segment 5 (TMS 5). Alanine scanning of all the amino acids of the TMS 5 by site directed
mutagenesis highlighted the importance of the motif as well as other residues of TMS 5, in drug
transport. The mutant variants of TMS 5 were placed in four different categories. The first
category had four residues G244, G251, G255 and G259, which are part of the conserved ‘motif
C’ and their substitution with alanine, resulted in increased sensitivity to drugs and displayed
impaired efflux of drugs. Interestingly, first category mutants, when replaced with leucine resulted
in more dramatic loss of drug resistance and efflux. Notwithstanding the location in core motif, the
second category included residues which are part of motif such as P260 and those which were not
part of motif such as L245, W248, P256 and F262, whose substitution with alanine, resulted in a
severe loss of drug resistance and efflux. Third category included G263, which although is a part
of the ‘motif C’, but unlike other conserved glycines, its replacement with alanine or leucine,
showed no change in the phenotype. The replacement of the remaining eleven residues of fourth
category did not result in any change. The putative helical wheel projection show clustering of
functionally critical residues to one side and thus suggests an asymmetric nature of TMS 5.




                                                124
P68B
Identification of CgPDR1 mutations involved in azole resistance among clinical Candida
glabrata isolates
Sélène Ferrari and Dominique Sanglard
Institute of Microbiology, University Hospital (CHUV), Rue du Bugnon 48, Lausanne 1011,
SWITZERLAND, Phone: +41 21 314 40 61, FAX: +41 21 314 40 60, e-
mail: selene.ferrari@chuv.ch

CgPdr1p is a C. glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-
transporter genes CgCDR1, CgCDR2 and CgSNQ2. Single point mutations in CgPDR1 have been
previously shown to increase the expression of at least CgCDR1 and CgCDR2 and thus contribute
to azole resistance. In this study, we investigated the incidence of CgPdr1p mutations in a
collection of C. glabrata clinical isolates. CgPDR1 was cloned and sequenced from 24 matched
pairs of azole-susceptible (MIC fluco. of 16 ug/ml or less) and azole-resistant (MIC fluco. of 32
ug/ml or more) isolates upregulating CgCDR genes. By comparison of CgPDR1 alleles from
azole-susceptible and -resistant matched isolates, we identified 19 distinct CgPDR1 alleles each
with a single amino acid substitution, which might confer hyperactivity to CgPdr1p in order to
mediate high expression of CgCDR genes. Moreover, the analysis of the CgPDR1 sequences of 56
unrelated azole-resistant clinical isolates allowed to characterize 36 additional putative gain-of-
function mutations. These 55 mutations were located at 49 distinct locations along the protein, and
encompassed three distinct protein domains: i) the region homologous to the inhibitory domain of
S. cerevisiae Pdr1p ii) the middle homology region and iii) the putative transcriptional activation
domain. Disruption of CgPDR1 in an azole-resistant isolate led to a drastic increase of azole
susceptibility and to the loss of CgCDR genes expression. Expression of some of the mutant
CgPDR1 alleles in the background of a CgPDR1-deleted strain confirmed the involvement of the
identified CgPdr1p amino acid substitutions in ABC-transporter genes upregulation and thus in
azole resistance. Interestingly, although CgCDR1, CgCDR2 and CgSNQ2 promoters contain
consensus binding sites for CgPdr1p, they are not always coordinately expressed in azole-resistant
isolates indicating that these genes might be diffentially regulated. In conclusion, this work
allowed to identify new gain-of-function CgPDR1 mutations responsible for CgCDR genes
constitutive upregulation and for azole resistance in several clinical C. glabrata isolates. Our
results demonstrate the high diversity of CgPDR1 mutations potentially involved in azole
resistance. Additional studies are now undertaken to understand how CgPdr1p gain-of-function
mutations affect transcription of the ABC-transporter genes.




                                               125
P69C
Mutations in drug resistance genes coupled with chromosome 5 rearrangements mediate
antifungal resistance in C. albicans
Alix Coste1, Francoise Ischer1, Anna Selmecki2, Anja Forche2, Judith Berman2 and
Dominique Sanglard1
1
  Microbiology Institute, University Hospital Lausanne, rue du Bugnon 48, Lausanne 1007,
Switzerland, Phone: + 41 (0)21 314 40 61 or 51, FAX: +41 (0)21 314 40 60, e-
mail: alix.coste@chuv.ch 2 Departments of Genetics, Cell Biology and Development, and
Microbiology, University of Minnesota, Minneapolis, Minnesota 55455

Azole resistance in C. albicans can be mediated by the ABC-transporters Cdr1p and Cdr2p. Both
genes are regulated by a trans-acting factor called Tac1p (for Transcriptional Activator of CDR
genes). TAC1 is located on Chr. 5 close to the MTL locus. TAC1 alleles exist as wild-type and
hyperactive alleles. Wild-type ones drive the high expression of CDR1/2 by the addition of
inducers, while hyperactive are responsible for constitutive high expression of CDR1/2. We first
showed that hyperactivity of TAC1 was due to acquisition of gain-of-function (GOF) point
mutations. We next demonstrated that high azole resistance levels were achieved when C. albicans
only exhibit hyperactive alleles, which can be obtained by loss of heterozygosis (LOH) at the
TAC1 locus on Chr. 5. In this work, we performed a study on 5 groups of further related isolates
containing azole-susceptible and -resistant strains and analysed their TAC1 alleles. Seventeen new
TAC1 alleles were isolated, including 10 wild-type and 7 hyperactive alleles. Five separate GOF
mutations responsible for TAC1 hyperactivity were identified. SNPs analysis of Chr. 5 determined
by which mechanism azole-resistant strains acquire only TAC1 hyperactive alleles: 1)TAC1 LOH
achieved either by mitotic recombination of a region flanking the TAC1 locus (2 cases) or within
the TAC1 locus (1 case) or entire Chr. 5 loss and duplication (1 case); 2)Acquisition of an
additional second GOF mutation in the remaining wild-type TAC1 allele (1 case). Moreover, CGH
and CHEF analysis performed on related azole-resistant strains identical for Chr. 5 SNPs analysis
and TAC1 alleles, revealed the presence of an iso-chromosome (5iL) in the most azole-resistant
strain (DSY735 and DSY289). This (5iL) leads to an increased copy number of azole-resistance
genes present on the 5L arm of Chr. 5, among which TAC1 and ERG11. In this study we
demonstrated for the first time that the development of resistance was not only due to the presence
of specific mutation(s) in azole resistance genes (ERG11 and TAC1) but also to their increase in
copy number by LOH events and addition of extra chromosome copies. This study shows that C.
albicans has the capacity to respond to antifungal agents by playing with separate strategies
involving single genes alteration or whole genome modifications.




                                               126
P70A
Assessment of mutated alleles activity of TAC1, a Candida albicans transcription factor
regulating drug resistance genes, using a one-hybrid system
Jerôme Crittin, Alix Coste, Françoise Ischer and Dominique Sanglard
Microbiology Institute, University Hospital Lausanne, rue du Bugnon 48, Lausanne 1011,
Switzerland, Phone: + 41 (0)21 314 40 61 or 51, FAX: +41 (0)21 314 40 60, e-
mail: alix.coste@chuv.ch

Azole resistance in C. albicans can be mediated by the ABC-transporter genes CDR1 and
CDR2. Both genes are regulated by a trans-acting factor called Tac1p (for Transcriptional
Activator of CDR genes). TAC1 alleles exist as wild-type and hyperactive alleles. Wild-type
alleles drive the high expression of CDR1/2 by the addition of inducers, while hyperactive
alleles are responsible for constitutive high expression of CDR1/2. Hyperactivity of TAC1 was
shown to be due to gain-of-function (GOF) point mutations. In order to develop a collection of
hyperactive TAC1 alleles and to determine regions important for TAC1 hyperactivity, a random
mutagenesis of TAC1 was performed. To screen all obtained mutated alleles, a simple and
rapid screening method was developed. A one-hybrid system was designed based on the
recovery of histidine prototrophy. Briefly, the HIS3 ORF was placed under the control of the
CDR2 promoter and the chimeric CDR2-HIS3 construction was introduced in a strain lacking
HIS3 (the reporter strain) and deleted for TAC1. Since the CDR2 promoter shows no basal
activity, the reporter strain was unable to grow on a minimal medium lacking histidine but only
in the presence of oestradiol or fluphenazine and of TAC1. In contrast, hyperactive TAC1
alleles (TAC1-5 and TAC1-7) restored growth in minimal medium without drug addition,
which is consistent with the hyperactive property of these alleles. This system was used for the
screening of the library of random mutagenized wild-type TAC1 alleles in order to determine
GOF mutations. The screening of around 3000 different colonies enabled the selection of 29
hyperactive TAC1 alleles from which 9 different GOF mutations (T225A, W239L, A736T,
A736V, I794V, N972D, N972I, N977D and I255stop) were identified. The majority of these
mutations were already identified in TAC1 alleles of clinical isolates, thus suggesting that GOF
mutations can occur in a limited number of positions in Tac1p. The one-hybrid system
developed here is rapid and powerful, and will be used in the development and characterization
of additional TAC1 alleles useful for the diagnostic of azole resistance in C. albicans.




                                               127
P71B
Minimal inhibitory concentration of fluconazol for planktonic and biofilm cells of oral
Candida species
Mariana Henriques, Ana Paula Ribeiro, Margarida Martins, Rosário Oliveira and
Joana Azeredo
IBB-CEB, University of Minho, Campus Gualtar, Braga 4710-057, PORTUGAL,
Phone: +351253604408, FAX: +351253678986, e-mail: mcrh@deb.uminho.pt

Non-Candida albicans Candida (NCAC) species are emerging as fungal pathogens causing
Candidiasis. Fluconazole is commonly used in antifungal therapy in doses determined according
to the susceptibility of Candida albicans grown in suspension. However, it is well known that, in
order to colonize and infect, microbial cells form biofilms. Thus, it is of utmost importance to
determine the susceptibility of the NCAC species, to antifungal agents, in both planktonic and
biofilm forms. Several clinical isolates were obtained from the oral cavity of patients with
Candidiasis. The isolates were identified using CHROMagar Candida and some were selected,
along with one Candida albicans reference strain, to be used in the determination of the minimal
inhibitory concentration (MIC) of fluconazole. It was also added to the assay one Candida
albicans reference strain. MIC was determined when 50% of cell death was obtained in both
planktonic and biofilm cells. In the former, MIC was measured according to the NCCLS standard
(by optical density at 620 nm) and to cellular activity (using XTT). MIC, of 48h biofilms, was
determined by the quantification of the total biomass (using crystal violet) and the activity
(XTT). Over 50% of the isolates, in a total of 71, presented green colour. Violet, white, blue and
pink colonies were also identified, confirming the presence of Candida albicans, Candida
glabrata, Candida tropicalis and Candida parapsilosis, respectively, on the oral cavity of infected
patients. The 8 clinical isolates selected for MIC determination, with one exception, presented
lower MIC values than the reference strain. It was interesting to notice that isolates, belonging to
the same species, displayed different MIC values. As it was expected, MIC values obtained for
biofilm cells were high.




                                                128
P72C
Antifungal activity of azoles, amphotericin B, 5-fluorocytosine and echinocandins on cell
surface hydrophobicity and in vitro model of Candida glabrata biofilm formation.
Sona Kucharikova1, Magdalena Lisalova2 and Helena Bujdakova1
1
  Microbiology and Virology, Faculty of Natural Sciences, Comenius University, Mlynska
dolina B-2, Bratislava SR 84215, Slovakia, Phone: 00421 2 60296636, FAX: 00421 2
65429064, e-mail: sonakucharikova@yahoo.com, Web: - 2 HPL spol. s.r.o, Laboratory of
Clinical Microbiology, Istrijska 20, 841 07 Bratislava

This study was focused on formation of biofilm in vitro and its association with the cell surface
hydrophobicity (CSH) and resistance to antifugal agents in clinical isolates of Candida
glabrata. The ERG11 gene expression during different phases of biofilm was investigated. The
tested collection included 50 C. glabrata clinical isolates as well as collection strain C. glabrata
ATCC 2001. For all isolates, MIC 95 to fluconazole (FLU), itraconazole (ITR), voriconazole
(VOR), amphotericin B (AMB), 5-fluorocytosine (5-FC), micafungin (MIC) and caspofugin
(CAS) was established by NCCLS M27-A2. The MIC 95 values were used to calculate
subinhibitory concentrations for experiments with biofilm and CSH. Biofilm was quantified by
the XTT reduction assay. The CSHs were determined by biphasic separation, percentage of
hydrophobic cells was calculated. The expression of ERG11 gene during biofilm formation
was established by the reverse-transcriptional PCR&#894; RNA was isolated from C. glabrata
ATCC 2001: after 1.5, 6, 24 and 48 h. Biofilm formed in presence and absence of FLU was
explored using confocal scanning laser microscopy (CSLM). Among 50 C. glabrata, resistance
was determined: to FLU (30%)&#894; ITR (100%)&#894; VOR and AMB (per 16%). All
isolates were susceptible to 5-FC, MIC and CAS. Biofilm showed to be weak in 10 isolates
(OD 490 <0.1), moderate in 18 isolates (OD 490 =0.1-0.3), relatively strong in 22 isolates and
collection strain (OD 490 >0.3). The 32 isolates manifested hydrophobic cells under 10%. For
12 clinical isolates, CSHs were determined between 10% to 30% and 7 isolates had
CSHs>30%. Isolates with strong and weak ability to form biofilm were tested for biofilm and
the changes in CSH in the presence of antifungals. Individual antifungal agents reduced CSH
significantly (p<0,005) with exception for CAS (p>0,005). The subinhibitory concentrations of
azoles (FLU, VOR) decreased the ability to form biofilm (p<0,005) with exception for ITR
(p>0,005). Moreover, agents moderately reduced biofilm formation in azole-resistant clinical
isolates. The AMB, 5-FC, MIC and CAS significantly reduced both CSH and biofilm
formation. The expression of ERG11 gene in C. glabrata ATCC 2001 decreased in maturation
phase of biofilm regardless of FLU presence. These results correlated with an observation of
biofilm by CSLM, documenting thinner biofilm formed in presence of FLU.




                                                 129
P73A
The development of Candida albicans biofilm: the ERG11 gene expression pattern
associated with a composition of different growth media.
Anna Kolecka1, Sona Kucharikova1, Dusan Chorvat2, Juraj Gasperik3 and Helena Bujdakova1
1
  Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius
University, Mlynska dolina B2, Bratislava SR 84215, Slovakia, Phone: +421 2 602 96 636,
FAX: +421 2 654 29 064, e-mail: kolecka@fns.uniba.sk, Web: - 2 International Laser Center,
Bratislava, Slovakia 3 Institute of Molecular Biology SAS, Bratislava, Slovakia

Purpose of this study was to investigate changes of C. albicans biofilm using two media: YNB
with amino acids and RPMI 1640, with non adjusted pH (about 5) and with adjusted pH to 7.0,
supplemented with 0.9% or 2.0% of glucose. Isolate C. albicans S11 and standard strain C.
albicans SC 5314 were used in experiments. Biofilm was quantified as the ability to adhere to
polystyrene plastic surface using XTT reduction assay and by the measurement of dry weight
of every sample. Majority of experiments were prepared also in the presence of 0.5 x MIC95 of
fluconazole (FLC), determinated for both tested strains. Additionally, the changes of the
ERG11 gene expression under different cultivation conditions were followed by reverse-
transcriptional PCR (RTPCR). The structure of 48-h biofilm was observed by confocal
scanning laser microscopy (CSLM). At standard conditions, (YNB+0.9% of glucose, pH non-
adjusted) C. albicans S11 proved a strong production of biofilm in comparison with standard
strain C. albicans SC 5314. The use of RPMI medium for biofilm formation (instead of YNB)
radically increased a production of biofilm in C. albicans S11, thus a correct measurement at
spectrophotometer was problematic, especially, when pH of media was 7.0. YNB medium with
pH=7.0 significantly stimulated the growth of biofilm created by both strains as well. The
influence of glucose concentration showed to be strain, media and pH dependent. The
efficiency of FLC to biofilm formation was tested in YNB+0.9% glucose, pH=7.0. In spite of
FLC resistance in C. albicans S11 this agent reduced biofilm formation in both FLC resistant
and susceptible strains. For RT PCR, four time points (1.5h, 6h, 24h, 48h) during biofilm
formation were selected for collection of RNA. Biofilm was prepared in the presence and
absence of 0.5 x MIC95 of FLC. While the presence of FLC in YNB+0.9% glucose with non
adjusted pH did not significantly affect ERG11 expression, upregulation of these gene was
proved in the same medium with FLC when pH=7.0. Profile of biofilm formed by both
Candida observed by CSLM confirmed the results obtained from the XTT reduction assay.
Presented results suggested a key role of cultivation conditions used for biofilm formation.
Moreover, these circumstances, especially neutral pH, preserved during formation of biofilm
can significantly affect the efficiency of FLC to C. albicans biofilm.




                                              130
P74B
Development and application of a two-hybrid system for the use in the human pathogenic
fungus Candida albicans
Bram Stynen and Patrick Van Dijck
VIB - Laboratory of Molecular Cell Biology, Catholic University of Leuven, Kasteelpark
Arenberg 31, Leuven-Heverlee 3001, Belgium, Phone: +32 (0)16 32 19 50, FAX: +32 (0)16 32
19 79, e-mail: bram.stynen@bio.kuleuven.be, Web: http://bio.kuleuven.be/mcb

The research on the human pathogenic fungus Candida albicans has some major restricions,
for example C. albicans has a diploid genome and lacks a sexual cycle. Moreover, C. albicans
has an alternative codon usage in which the codon CUG is translated into serine and not into
leucine like the majority of organisms. Because of this alternative codon usage, the application
of the yeast two-hybrid system for screening genes from C. albicans is not optimal. Many C.
albicans proteins expressed in yeast will have a wrong amino acid sequence and this will
influence the interactions observed in a screening. The development of a two-hybrid system for
the use in C. albicans itself overcomes this problem. Components of this two-hybrid system
will have to be functional in C. albicans, like LexA from Staphylococcus aureus as DNA
binding protein and LacZ from Streptococcus thermophilus as reporter protein (Russell and
Brown, 2005; Uhl and Johnson, 2001). We will try out different strategies, like changing the
promoters, activation domains and reporter genes to provide an optimal system that can be
used as a better alternative to the existing yeast two-hybrid system. This new technology
should be useful both for the confirmation of expected interactions as for the discovery of new
interactions by screening with a gDNA/cDNA library. After finishing the development we will
apply the C. albicans two-hybrid system looking for interacting partners of the signal
transduction proteins Ras1 and Cdc25. Both proteins will be inserted separately into the bait
plasmid. A prey gDNA library will be used to screen for interaction partners. Russell, C.L.,
Brown, A.J. (2005). Expression of one-hybrid fusions with Staphylococcus aureus lexA in
Candida albicans confirms that Nrg1 is a transcriptional repressor and that Gcn4 is a
transcriptional activator. Fungal Genet. Biol., 42(8):676-683. Uhl, M.A., Johnson, A.D.
(2001). Development of Streptococcus thermophilus lacZ as a reporter gene for Candida
albicans. Microbiology, 147(5):1189-1195.




                                               131
P75C
The preparation of suitable methods for study of the secretion pathway of proteinase
Sapp1p of the pathogenic yeast Candida parapsilosis
Zuzana Vinterova1, Jiri Dostal1, Hana Flegelova2, Iva Pichova1, Hana Sychrova2 and
Olga Hruskova-Heidingsfeldova1
1
  Gilead Sciences Research Centre, Institute of Organic Chemistry and Biochemistry AS CR,
Flemingovo nam. 2, Prague 16610, Czech Republic, Phone: +42(0)220183242,
FAX: +42(0)220183556, e-mail: vinterova@uochb.cas.cz 2 Institute of Physiology AS CR,
Videnska 1083, Prague, 14220, Czech Republic

The yeast Candida parapsilosis is an opportunistic pathogen. One of the most important
virulence factors of the pathogenic Candida spp. is the production of secreted aspartic
proteinases (Saps). C. parapsilosis produces at least two Saps, named Sapp1p and Sapp2p.
These proteinases are synthesized as pre-pro-enzymes, however, only mature proteinases were
detected in the growth media. The site of the zymogen processing remains unknown. The long-
term aim of this work is the description of the Sapp1p secretory pathway and elucidation of
where and when the pro-Sapp1p is activated. Development of suitable methods was an
essential prerequisite for further studies. We prepared three different types of rabbit polyclonal
antibodies directed against (a) putative signal sequence, (b) the end of pro-peptide and (c) a
unique part of the mature Sapp1p sequence. We optimized the procedure of removal of the
unspecifically bound Sapp1p from the cell surface, using wash buffers with various detergents
or reducing agents. The enzyme activity assays confirmed that the fraction of Sapp1p
integrated to the cell wall is proteolytically active. We also optimized methods for the yeast
cell disintegration. We tested several methods including mechanical (X-press, Bio-Neb
disintegrator, glass beads) and chemical approaches (forming of protoplasts and their lysis).
The disintegrated cells were then partially fractionated and the presence of mature Sapp1p and
its precursors in the individual fractions was analyzed using the Western blotting. Another
approach to analyze the Sapp1p secretory pathway employed Saccharomyces cerevisiae. The
S. cerevisiae cells were transformed with various plasmids containing the SAPP1 gene. We
also used the SAPP1 gene tagged with GFP on N- or C- terminus. The expression of Sapp1p
was examined using fluorescent microscopy and Western blotting. The results suggest that in
S. cerevisiae the GFP-tagged Sapp1p is targeted to Golgi. This work was supported by the
grants of the Czech Science Foundation (No. 203/05/0038) and of the Ministry of Education
CR (No. LC531).




                                                132
P76A
Elucidation of additional components of the Candida albicans calcineurin signaling
cascade
Jennifer Reedy1, Scott Filler2 and Joseph Heitman1
1
  Molecular Genetics and Microbiology, Duke University, 320 CARL Bldg, Research Drive,
Box 3546, Durham NC 27710, USA, Phone: +001 919 684 3036, FAX: +001 919 684 5458, e-
mail: reedy004@mc.duke.edu 2 Division of Infectious Diseases, Los Angeles Biomedical
Research Institute, Harbor-UCLA Medical Center

The serine-threonine specific calcium-calmodulin activated protein phosphatase calcineurin plays
a crucial role in the virulence of two human fungal pathogens, Cryptococcus neoformans and
Candida albicans, via distinct mechanisms. Calcineurin is essential for C. neoformans growth at
high temperature, whereas C. albicans calcineurin promotes tolerance to azoles, and survival
during the calcium stress imposed by growth in serum. Our studies focus on elucidating the roles
of calcineurin in virulence of C. albicans and the identification of other components of the
calcineurin signaling pathway via candidate gene disruption and screening of homozygous and
heterozygous mutant libraries. The RCAN (Regulator of Calcineurin) family of proteins consists
of calcineurin binding proteins that modulate calcineurin activity in S. cerevisiae, C. neoformans,
and mammalian cells. A member of this family in C. albicans, RCN1, was identified based upon
the conserved FLSPPxSPP domain, and the RCN1 gene was disrupted using the SAT1 flipper
cassette. rcn1/rcn1 mutant strains exhibit sensitivity to LiCl, fluconazole, and SDS, but have
wildtype tolerance to serum and calcium. Additionally, heterologous overexpression of C.
albicans Rcn1 in S. cerevisiae inhibits S. cerevisiae calcineurin and rescues the calcium sensitivity
of a pmc1 deletion strain. Preliminary results suggest that rcn1/rcn1 strains, unlike calcineurin
mutants strain, have wildtype virulence in a murine systemic candidiasis model. Interestingly, in
an oropharyngeal candidiasis model neither the rcn1/rcn1 nor the cnb1/cnb1 deletion mutants
have a virulence defect. This further supports previous findings that the role of calcineurin in
virulence is host niche specific. We have also identified MID1 and CCH1, which encode two
components of a cell membrane calcium channel important for calcineurin activation in S.
cerevisiae. Deletion of either MID1 or CCH1 results in hypersensitivity to membrane stress (SDS
or fluconazole), and LiCl, but mid1/mid1 and cch1/cch1 mutants have wildtype sensitivity towards
high calcium and serum. We are further probing the sensitivities of mid1/mid1 cch1/cch1 double
mutants and the patterns of expression of these genes in response to calcium and calcineurin
signaling.




                                                133
P77B
Tetracycline-inducible expression of secreted aspartyl proteases in Candida albicans
allows isoenzyme-specific inhibitor screening
Peter Staib1, Björn Degel2, Julia Blass-Warmuth3, Ulrich Lermann3, Reinhard Würzner4,
Michel Monod1, Tanja Schirmeister2 and Joachim Morschhäuser3
1
  Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, Avenue de Beaumont 29,
Lausanne 1011, Switzerland, Phone: +41 (0) 21 31 46876, FAX: +41 (0) 21 31 40378, e-
mail: Peter.Staib@chuv.ch 2 University of Würzburg, Institute for Pharmacy and Food
Chemistry, Würzburg, Germany 3 University of Würzburg, Institute for Molecular Infection
Biology, Würzburg, Germany 4 University of Innsbruck, Department of Hygiene,
Microbiology and Social Medicine, Innsbruck, Austria

A new approach to treat C. albicans infections is to use major virulence determinants of C.
albicans as drug targets. C. albicans possesses a gene family encoding secreted aspartic proteases
(Sap1-Sap10), and these enzymes have been linked with the virulence of the fungus since their
discovery. Proposed functions during infection include digestion of host proteins for nutrient
supply, adherence to and invasion of host tissues, and the evasion of host defense mechanisms.
Because different Sap isoenzymes are expected to play distinct roles during infection, Sap
inhibitors should be active against all or at least many of the ten known members of the Sap
family. In a growth medium containing protein, e.g., bovine serum albumin (BSA), as the sole
source of nitrogen, C. albicans specifically expresses the Sap2 isoenzyme, which allows growth
by BSA degradation. Consequently, a C. albicans sap2 deletion mutant can not grow under these
conditions. Using a tetracycline-inducible gene expression system we succeeded to induce eight
single isogenes of the SAP family in a sap2 mutant of the wild-type strain SC5314 to enable
growth of the cells on BSA by addition of doxycycline to the growth medium. The activity of
potential protease inhibitors against the different Sap members can therefore easily be determined
by their ability to block growth of the corresponding strains in this medium. Expression of specific
Sap isoenzymes also allows purification of the enzymes from the supernatant of the corresponding
strains for detailed inhibitor studies. In addition, doxycycline-inducible SAP gene expression gives
the opportunity to further characterise the role of individual Saps of C. albicans in in vitro models,
e.g. during tissue adhesion or invasion.




                                                 134
P78C
Functional analysis of Pneumocystis carinii genes in Schizosaccharomyces pombe
Moira Cockell1, Philippe Hauser1, Libera Lo Presti1, Lorenzo Cerutti2 and Viesturs Simanis3
1
  Institute of Microbiology, CHUV, Rue du Bugnon 48, Lausanne VD CH-1011, Switzerland,
Phone: +41 (0)21 314 4084, FAX: +41 (0)21 314 4060, e-mail: moira.cockell@hospvd.ch,
Web: http://www.chuv.ch/imul/imu_home/imu_recherche/imu_recherche_hauser.ht 2 Swiss
Institute of Bioinformatics, Batiment Genopode, Lausanne 3 ISREC/ EPFL Faculty of Life
Sciences, Lausanne

The absence of methods to culture Pneumocystis species in vitro has rendered their biology
difficult to study. As a consequence, few effective targets for the treatment of opportunistic
infection by the human pathogen P. jirovecii have been identified to date. However, the
phylogenetic pedigree of Pneumocystis species, suggests they are likely to employ conserved
fungal-specific proteins and pathways that are essential for viability. Identification of such
modules may uncover novel targets for directed pharmaceutical intervention. To this end, we are
assessing the ability of sequenced genes from the rat pathogen P. carinii to complement the loss of
essential functions in a collection of deletion mutants from the close evolutionary neighbour of
Pneumocystis, Schizosaccharomyces pombe. In the first phase of our study, we have used
bioinformatics to identify a subset of P. carinii ESTs with low homology to vertebrate genes and
significant homology to S. pombe genes with undetermined function. As a second step, the
phenotypes conferred by deletion of the corresponding S. pombe genes is assessed and deletions
giving rise to inviable cells are retained for analysis by complementation. Using this approach, we
have determined that haploid cells bearing a deletion of the S. pombe trf1 gene are inviable, while
diploid cells heterozygote for trf1 delta have no apparent defect. Loss of trf1 function is rescued
by ectopic expression of either the S. pombe Trf1 protein or its P. carinii orthologue, but not
complemented by the more distant S. cerevisiae homologue, Tbf1p. Nevertheless, the presence of
a related DNA-binding motif in S. cerevisiae Tbf1p, S. pombe Trf1p and P. carinii Trf1p, raises
the possibility that each protein has a similar role. Previous studies indicate that S. cerevisiae
Tbf1p binds to sub-telomeric and other regions of the chromosomes, but have not addressed its
essential cellular function. Our preliminary data suggest that S.pombe Trf1p is also predominantly
localised in the non-rDNA compartment of the nucleus and is required for accurate chomosome
segregation during vegetative growth. Given that the subtelomeres of P. carinii harbour the
repeated MSG gene family whose recombination is important for antigenic variation and
resistance to host immune responses, and that S. pombe cannot tolerate chromosome aneuploidy,
the P. jirovecii Trf1p homologue may prove to be a promising candidate for the development of
drugs which interfere with its function.




                                               135
P79A
The transcription factor MRR1 controls expression of the MDR1 efflux pump and
mediates multidrug resistance in Candida albicans
Joachim Morschhäuser1, Katherine S. Barker2, Teresa Liu2, Ramin Homayouni2, Julia Blaß-
Warmuth1 and P. David Rogers2
1
  Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg
97070,         Germany,       Phone: ++49-931-312152,         FAX: ++49-931-312578,       e-
mail: joachim.morschhaeuser@mail.uni-wuerzburg.de 2 University of Tennessee Health
Science Center, Memphis, Tennessee, USA

Constitutive overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the
major facilitator superfamily, is a frequent cause of resistance to fluconazole and other toxic
compounds in clinical Candida albicans strains, but the mechanism of MDR1 upregulation has not
been resolved. By genome-wide gene expression analysis we have identified a zinc cluster
transcription factor, designated as MRR1 (multidrug resistance regulator), that was coordinately
upregulated with MDR1 in drug-resistant, clinical C. albicans isolates. Inactivation of MRR1 in
two such drug-resistant isolates abolished both MDR1 expression and multidrug resistance.
Sequence analysis of the MRR1 alleles of two matched drug-sensitive and drug-resistant C.
albicans isolate pairs showed that the resistant isolates had become homozygous for MRR1 alleles
containing single nucleotide substitutions that caused amino acid exchanges in the encoded
protein. Introduction of these mutated alleles into a drug-susceptible C. albicans strain resulted in
constitutive MDR1 overexpression and multidrug resistance. Therefore, we have identified the
central regulator of MDR1 expression in C. albicans and elucidated the molecular basis of a major
mechanism of multidrug resistance development in clinical C. albicans strains.




                                                136
P80B
Accessing Candida albicans gene and genomic information at Candida Genome Database
Maria C. Costanzo, Marek S. Skrzypek, Martha B. Arnaud, Prachi Shah, Gail Binkley, Stuart
R. Miyasato, Kathy Kegang Zhu and Gavin Sherlock
Genetics, Stanford University School of Medicine, Stanford University, Stanford CA 94305,
USA, Phone: 1-650-498-6012, FAX: 1-650-724-3701, e-mail: maria@genome.stanford.edu,
Web: http://www.candidagenome.org/

The Candida Genome Database (CGD, http://www.candidagenome.org/) is a freely available
online resource containing curated gene, protein, and genome information for the opportunistic
fungal pathogen Candida albicans. The CGD team performs manual curation of the literature
to collect C. albicans gene names and aliases; assign Gene Ontology (GO) terms describing the
molecular function, biological process, and subcellular localization of each gene product;
curate mutant phenotypes; and write free-text description lines that summarize the function and
biological context of each gene product. Each piece of information is linked to a reference, and
each gene is linked to a list of the publications in which the gene has been characterized. All of
this information is displayed on, or linked from, a CGD Locus page for each gene or
chromosomal feature.
CGD also maintains and displays the genomic sequence. Most recently, the chromosome-level
genome assembly, Assembly 20, was added to the already widely used Assemblies 4, 5, 6, and
19. In order to facilitate viewing and browsing through versions of the C. albicans genome and
tracing sequence-based data between the different assemblies, CGD implemented GBrowse, an
open-source genome browser developed as part of the Generic Model Organism Database
project (http://www.gmod.org/). The two most complete versions of the C. albicans genome,
Assemblies 19 and 20, are accessible in GBrowse and can be navigated independently of each
other. The tool displays a user-configurable set of Tracks, each showing a class of features
such as known or predicted ORFs, tRNAs, centromeres, or contigs. Sequence-based mappings
of the contigs from Assemblies 4, 6, and 19 and the ORFs from Assemblies 6 and 19 onto the
newer assemblies, are visually represented in GBrowse as the Historic Assemblies tracks. In
addition, CGD provides a BLAST tool that allows searching through sequences from
Assemblies 4, 5, 6, 19, and 20. The genomic context of the BLAST results may be viewed in
GBrowse and any selected region of DNA or protein sequence may be downloaded from the
browser for further analysis.
Additionally, CGD provides community resources including a gene name registry, a searchable
colleague and laboratories database, a list of relevant meetings, and a job opportunities web page.
CGD curators welcome comments and suggestions by email, at candida-
curator@genome.stanford.edu. CGD is supported by grant RO1 DE15873 from the NIDCR at the
NIH.




                                               137
P81C
Identification of covalently linked cell wall proteins in Candida albicans: A comparative
approach
Ekkehard Hiller1, Alfred Nordheim2, Herwig Brunner1 and Steffen Rupp1
1
  , Fraunhofer IGB, Nobelstrasse 12, Stuttgart 70569, Germany, Phone: +49 (0)711 970 4050,
FAX: +49 (0)711 970 4200, e-mail: hiller@igb.fraunhofer.de, Web: http://www.igb.fhg.de 2
Institute for Cell Biology, Department of Molecular Biology, University of Tuebingen, Auf der
Morgenstelle 15, 72076 Tuebingen, Germany

Candida albicans is a common human pathogen which may cause superficial and systemic
infections especially in persons with weakened immune system. During infection, the cell wall and
its components plays an important role because this complex network built of glucan, chitin,
mannan and proteins is the interface between host and pathogen. It was shown that several of the
embedded proteins are crucial for adhesion and interaction with the host. During yeast to hyphal
transition significant changes in the cell wall proteome were first suggested by microarray
experiments and further confirmed by studies focusing on cell wall proteomics. In our work, we
compare the results of different approaches for solubilisation of covalently bound proteins or
peptides thereof from isolated cell walls for identification with MS/MS. Trypsin, endoproteinase
Glu-C and cyanogene bromide alone or in combination were used, and the effects of a pre-
treatment of the cell wall with a beta-1,3 glucanase tested and compared to each other. Two
different types of mass spectrometers were used for identification of the peptides. Overall, 33
proteins were identified in yeast growth form, 6 with a predicted GPI-anchor. In contrast, 14 of the
identified 18 proteins in hyphal cells carry this specific anchor sequence. Based on the level of
transcripts, 11 of these proteins are upregulated under hyphal inducing conditions. A large
variance in the identification of proteins and the number of peptides corresponding to one protein
depending on the method used was found.




                                                138
P82A
Antigenome technology, a novel approach for the development of fungal subunit vaccines
Petra Schlick, Alexander von Gabain, Eszter Nagy and Andreas Meinke
Antigen Discovery, Intercell AG, 6 Campus Vienna Biocenter, Vienna 1030, AUSTRIA,
Phone: +43 (0)1 20620 212, FAX: +43 (0)1 20620 805, e-mail: pschlick@intercell.com

Recently, we reported a novel screening procedure employing the host immune system to select
the best vaccine candidates against a particular disease (Etz et al., 2002). Briefly, serum antibodies
induced by pathogens in consequence of their interaction with the human host are used to select
candidate peptides displayed on the surface of E. coli via outer membrane proteins. Covering the
full genome of the pathogen, both uniformly small (linear epitopes) or uniformly medium-sized
peptides (possibly conformational epitopes) are displayed, to ensure that all potential antigens can
be identified. This technology, defining the “antigenome” of a pathogen, was first applied to
Staphylococcus aureus and has been extended to other important pathogenic bacteria such as
Streptococcus pneumoniae and Streptococcus pyogenes, and has been validated by rediscovering
the majority of previously known protective antigens. Furthermore, as the genomes are randomly
fragmented, the antigenome technology does not rely on genome annotation and, thus, has the
potential to select proteins that are not predicted by ORF finding algorithms. Pathogenic fungi
have emerged as major causes of human disease and the morbidity and mortality associated with
these infections are substantial. The most common cause of fungal infections are Candida spp.,
which are an increasingly important cause of disease among patients in intensive care units. Since
the antigenome approach is not dependent on the nature of genomic DNA, the technology might
serve as a potential means for the identification of antigens derived from these eukaryotic
pathogens. Using Candida albicans as a model system, we want to extend the antigenome
technology beyond bacterial targets to the development of fungal subunit vaccines and describe
the project outline in detail.

Etz, H. et al. (2002), PNAS 99, 6573-6578.




                                                 139
P83B
Systematic investigation of cell wall modulations in clinical isolates of Candida glabrata
Oliver Bader, Alexander Schwarz, Uwe Gross, Michael Weig and the EURESFUN
Consortium
Medical Microbiology, Georg-August-University Göttingen, Kreuzbergring 57, Göttingen
37075, Germany, Phone: +49 (0)551 39 2346, FAX: +49 (0)551 39 5861, e-
mail: obader@gwdg.de

The cell wall of pathogenic fungi is the interface to the host during colonization and infection
mediating a multitude of interactive processes. Even though it is long established that bacterial cell
walls can mediate drug resistance, this has not been looked at in fungi. In order to systematically
investigate resistance mechanisms in pathogenic Candida species, we have established a collection
(MycoLabNet-EU), containing over 400 fully or intermediate drug resistant clinical yeast isolates,
within the EURESFUN consortium. Over 100 Candida glabrata isolates out of this collection
were examined for drug efflux with rhodamine 6G by FACS analysis as well as tolerance to
several cell wall and other stresses. Azole cross-resistant isolates generally displayed high
efficiency drug efflux. Isolates with intermediate azole MIC90 values displayed only low to
medium drug efflux, but were more tolerant towards cell wall stresses. These isolates also were
less susceptible towards the echinocandin caspofungin. This may point towards compensational
modulations of the cell wall in response to antifungal drug treatment with azoles when increased
drug efflux is not available.




                                                 140
P84C
Identification of transcriptional activation targets of the transcription factor Tac1p
associated with azole resistance in clinical isolates of Candida albicans
Teresa Liu1,      Sadri Znaidi2,     Kathy Barker1,       Lijing Xu3,     Ramin Homayouni3,
                       4                   2                     1
Joachim Morschhäuser , Martine Raymond and P. David Rogers
1
  Clinical Pharmacy, University of Tennessee Health Science Center, 50 N. Dunlap St. WPT
Rm 304, Memphis TN 38103, USA, Phone: 901-287-5388, FAX: 901-287-5036, e-
mail: tliu@utmem.edu 2 Institute for Research in Immunology and Cancer, Montreal, Quebec,
Canada 3 University of Memphis, Memphis, Tennessee, USA 4 Institue für Molekulare
Infektionsbiologie, Universität Würzburg, Würzburg, Germany

Purpose: The transcription factor Tac1p has been shown to control the expression of the genes
encoding the azole resistance-conferring ABC-transporters Cdr1p and Cdr2p in C. albicans. The
purpose of this study was to identify additional transcriptional activation targets of Tac1p.

Methods: Four matched sets of clinical isolates originally obtained from patients who failed azole
therapy were used in this study. All 4 isolates with increased fluconazole MICs have previously
been shown to exhibit increased expression of CDR1 and CDR2 as compared to their matched
azole-susceptible isolates. SZY31 (tac1delta::FRT/tac1delta::FRT) was derived from clinical
isolate 5674 and SZY35 (tac1delta::FRT/tac1delta::FRT) was derived from strain SC5314. Drug
response experiments were conducted by exposing SC5314 and SZY35 to the IC50 of
ketoconazole (KCZ; 19.13 mcg/ml) for 3 hours. Total RNA was isolated and DNA microarray
analysis was performed. Gene expression profiles were compared between matched isolate pairs,
between isolates 5674 and SZY31, and between the drug exposure experiments. Chromatin
immunoprecipitation combined with DNA microarrays (ChIP-chip) were performed for a strain
expressing 3xHA-tagged Tac1p at its C-terminus and the parental untagged strain in order to
identify genes whose promoters were bound by Tac1p.

Results: A total of 43 genes were up-regulated in all 4 matched sets. Genes up-regulated included
CDR1, CDR2, ECM41, GPX1, HSP12, IFD4, IFU5, LCB4, NDH2, RTA3, IPF7530, and TAC1.
Of the 43 genes, 36 were found to be TAC1 dependent in 5674 when compared to SZY31. Of the
37 genes whose promoters were found to be bound in vivo by Tac1p-HA by ChIP-chip analysis,
11 were also found to be differentially expressed in the 4 matched isolate sets studied. These
included CDR1, CDR2, RTA3, IFU5, GPX1, and LCB4. Of the 859 genes up-regulated in response
to KCZ in SC5314, 49 genes were found to overlap with the matched sets data or the ChIP-chip
data. Twelve of these genes were found to be at least TAC1 partially dependent in response to
KCZ, including CDR1, CDR2, IFU5, LCB4, NDH2, and RTA3.

Conclusion: These results identify new transcriptional activation targets of Tac1p, including those
differentially expressed with CDR1 and CDR2 in association with azole resistance and suggest a
role for Tac1p in phospholipid and sterol metabolism.




                                               141
P85A
The activation mechanism of the transcription factor Upc2p in yeast
Chelsea Samaniego and Theodore White
Pathobiology, University of Washington/SBRI, 307 Westlake ave N, Seattle WA 98109, USA,
Phone: 206-256-7156, FAX: 206-256-7229, e-mail: chelsea.samaniego@sbri.org

The development of resistance to antifungal agents by pathogenic fungi is a considerable
clinical problem. Fungal ergosterol biosynthesis is the target of several antifungals including
azoles. The transcription factor Upc2p acts on genes in the ergosterol biosynthetic pathway and
has been shown to be involved in azole resistance in the human pathogen, Candida albicans
(Ca) and the yeast model system Saccharomyces cerevisiae (Sc). The single Ca protein
CaUpc2p shows significant homology to two Sc proteins, ScUpc2p and ScEcm22p. In silico
analysis of CaUpc2, ScUpc2p and ScEcm22p identifies an anchoring transmembrane domain,
a Zn(2)-Cys(6) binuclear cluster transcription factor domain and several nuclear localization
sequences (NLS). By analogy to sterol regulation in mammalian cells, we propose that the
transmembrane domain maintains inactive Upc2p in a membrane outside the nucleus. When
sterols are depleted, this localization is destabilized. Subsequently, specific proteases cleave
the N-terminal DNA binding domain, allowing the domain to translocate to the nucleus via
NLS sequences and activate specific genes. To test this, ScUpc2p-GFP fusions were examined
in Sc using fluorescence microscopy. The GFP tagged protein was localized both inside and
outside the nucleus of the Sc cells. No difference in localization was observed in the presence
of fluconazole, which is known to activate Upc2p. Staining with a series of vital dyes has ruled
out colocalization with several organelles but definitive colocalization has not been
established. The presence of dual transcription factors in Sc may complicate the analysis of
ScUpc2p. To address this, the ECM22 gene will be deleted in the ScUpc2p-GFP fusion strain.
Localization in Ca will be examined using a CaUpc2p-GFP fusion. A series of fluorescently
tagged CaUpc2p truncations are also being constructed to elucidate the roles of the various
protein domains in CaUpc2p localization. To determine whether proteolytic cleavage is
involved in Upc2p activation, immunoblots of TAP-tagged ScUpc2p have been done and
cleavage is being assessed based on size and location of proteins detected with antibodies to
the TAP-tag. A TAP-tagged CaUpc2p is also being constructed and will also be to detect
cleavage by immunoblot and to purify CaUpc2p for mass spectral analysis.




                                               142
P86B
Promoter analysis of the Candida albicans azole-inducible zinc cluster transcription
factor Upc2p
Samantha Hoot and Theodore White
Pathobiology, University of Washington/Seattle Biomedical, 307 Westlake Ave Suite 500,
Seattle WA 98109, USA, Phone: 4257501560, FAX: 2062567229, e-mail: sam.hoot@sbri.org

Treatment of Candida albicans (Ca) infections in immunocompromised patient populations is
complicated by the emergence of antifungal drug resistance. One major class of antifungal
drugs, the azoles, targets the biosynthesis of ergosterol, the primary sterol component of the
fungal cell membrane, by inhibiting the enzyme Erg11p. In addition to other mechanisms, the
regulation of ERG11 expression can play a central role in resistance to azoles. The
transcription factor Upc2p has recently been shown to play a role in the upregulation of
ergosterol biosynthetic genes including ERG11 in response to azoles. Evidence suggests that
Upc2p binds to the promoters of target genes via a conserved 7 base-pair sterol response
element (SRE). Interestingly, the promoter of CaUPC2 itself contains an SRE, suggesting that
expression of the transcription factor may be self-regulated. Promoter analysis has been
performed by creating a series of 100 bp deletions of the UPC2 promoter fused to the
luciferase reporter gene. These constructs have been tested for azole inducibility in both the
wild-type strain BWP17 as well as a delta upc2/delta upc2 strain. In the presence of azole
drugs, the full-length 750 base pair (bp) UPC2 promoter is induced 50-fold. Deletion analysis
suggests that two regions contribute to azole induction: the -450bp to -350bp region containing
the SRE and another region from -350bp to -250bp. When tested in the deltaupc2/delta upc2
strain, it was found that the promoter was still azole inducible, suggesting that Upc2p is not the
sole transcription factor controlling azole induced transcription from the UPC2 promoter. The
level of induction in the presence of azoles was 8-fold, and the inducible element appears to be
in the 350-250bp region. Mutation of the putative SRE has been performed, as well as finer
deletions in the 350-250bp region, and these constructs will be used to further confirm the
azole inducible element(s) within this promoter. In addition, 5’ RACE analysis has mapped the
start of transcription of UPC2 and has shown two distinct mRNAs, differing in the length of
the 5’UTR (-361bp and -150bp from translational start). The role of these two sites in
transcriptional and post-transcriptional regulation is being investigated. This work aims to
further characterize the role of azole inducible transcription of UPC2 its relationship to azole
drug resistance.




                                                143
P87C
Immunoproteomic analysis of the protective response obtained from vaccination with C.
albicans ecm33 mutant in mice
Raquel Martínez-López, Lucía Monteoliva Diez, Rosalía Diez-Orejas, Cesar Nombela and
Concha Gil García
Microbiología II, Facultad de Farmacia, UCM, Plaza de Ramón y Cajal S/N, Madrid 28040,
SPAIN, Phone: +34 91 394 17 55, FAX: +34 91 394 17 45, e-mail: raquelml@farm.ucm.es,
Web: http://www.ucm.es

Ecm33p is a widely distributed fungal protein with functional relevance; mainly related to cell
wall both in S. cerevisiae1 and C. albicans2. The C. albicans ecm33 mutant exhibits cell wall
defects with enhanced sensitivity to different compounds that interfere in cell wall components
polymerization and a sharp tendency to flocculate extensively. In addition, Ecm33p is required for
normal C. albicans yeast to hyphae transition, for virulence and for normal cell wall architecture
as well as normal function and expression of cell surface proteins in C. albicans2. Electronic
microscopy of S. cerevisiae and C. albicans ecm33 mutant cell walls revealed defects in the
organization of the glucan layer as well as an extended mannoprotein layer, which could be
responsible for the pleiotropic defects displayed by this mutant3. Vaccination of BALB/c mice
with 2,5x106 cells of C. albicans ecm33 mutant protected them from a subsequent infection with
the virulence strain SC5314 (1x106cells) in a murine model of systemic candidiasis, extending the
survival time of more than 85% of them from 5 to longer than 45 days. To our knowledge, this is
the most protective response reported so that, we have used an inmmunoproteomic approach in
order to find out the immunogenic proteins that could be good candidates for the development of a
non-cellular vaccine against systemic candidiasis. Western blot analysis with the sera extracted
from vaccinated mice with C. albicans ecm33 mutant and 2D gels of C. albicans proteins revealed
the presence of antibodies against 28 different proteins, some of them describe as immunogenic
for the first time. We have used different sera (obtained 15, 45 and 60 days post vaccination)
trying to cover the antibody response generated along the time and also different protein extract
(protoplast extracts composed by cytoplasmic proteins and SDS-DTT extracted proteins enriched
in surface proteins) in order to explore the antibody response against proteins of different cellular
compartments.

1. Pardo, M. et. al. (2004).Microbiology (150) p.4157.
2. Martinez-Lopez, R. et. al. (2004) Microbiology (150) p.3341.
3. Martinez-Lopez, R. et. al. (2006) Eukaryot. cell (Jan) p.140.

This work was supported by grants BIO 2006-01989 from the commission Interministerial de
Ciencia y Tecnología (CYCIT, Spain).




                                                144
P88A
Conditional inactivation of SUN genes in Candida albicans reveals an essential and
conserved cell-wall related function required at a late step of cell separation.
Arnaud Firon, Sylvie Aubert, Ismaïl Iraqui, Sophie Goyard, Guilhem Janbon and
Christophe d’Enfert
Fungal Biology and Pathogenicity, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015,
FRANCE, Phone: +33 (0)1 4568 8205, FAX: +33 (0)1 4568 8938, e-mail: afiron@pasteur.fr

SUN genes encodes ascomycete-specific proteins that share a signal peptide and a carboxy-
terminal domain containing a four cysteines motif predicted to bind iron. In S. cerevisiae, the 4
Sun proteins have been associated to various cellular processes although their precise function
remain unclear. Here, we report the characterization of the 2 C. albicans SUN genes : SUN41 and
SUN42. C. albicans strains with null mutations in the SUN41 or SUN42 genes were constructed.
Phenotypic analysis of these deletion mutants revealed that SUN41 is necessary for cell separation,
hyphal elongation and biofilm formation while inactivation of SUN42 results in minor phenotypic
alterations. Repeated attempts to obtain a C. albicans sun41 sun42 double mutant were
unsuccessful. Thus, conditional mutants with one SUN gene inactivated and the second placed
under the control of the repressible MET3 promoter were generated. Under repressible conditions,
the conditional double mutants showed a osmo-remediable lethal phenotype mainly due to mother
cell lysis following septation (both in yeast and hyphal conditions). These data indicate that C.
albicans Sun41p and Sun42p share a common essential cell wall related function required at a late
step of the cell separation process. Additional data suggest that the Sun41p and Sun42p perform
identical function but are differentially regulated at the transcriptional level. The essential function
of these proteins is conserved among yeasts since ectopic integration of S. cerevisiae orthologues
can suppress the conditional lethal phenotype associated to the inactivation of C. albicans SUN
genes. The conserved carboxy-terminal cysteine residues were shown to be critical for SUN
activity. The cell wall defect of conditional sun mutants result in a complex pattern of sensitivities
and resistance to a set of chemical agents acting on the cell wall or cell membranes (calcofluor
white, nikkomicin Z, caspofungin, amphotericin B and azoles). We are currently investigating a
probable enzymatic activity of the Sun proteins towards cell wall polymers that is suggested by
our data and those obtained by others in S. cerevisiae.




                                                 145
P89B
Prevalence of DHPS polymorphisms associated with sulfa resistance in South African
Pneumocystis jirovecii strains.
Leigh Dini1, Mignon du Plessis1, Michelle Wong2, Alan Karstaedt2, Victor Fernandez3 and
John Frean1
1
  Parasitology Reference Unit, National Institute for Communicable Diseases, 1 Modderfontein
Road, Sandringham 2192, South Africa, Phone: +27 (0) 11 555 0311, FAX: +27 (0) 11 555
0446, e-mail: leighd@nicd.ac.za, Web: www.nicd.ac.za 2 Chris Hani Baragwanath Hospital,
Johannesburg South Africa 3 Swedish Institute for Infectious Disease Control, Stockholm
Sweden

Drug resistance in Pneumocystis jirovecii has emerged as a possibility within the last decade.
Trimethoprim-sulfamethoxazole (TMP-SMX) is the drug of choice for treatment and prophylaxis
of Pneumocystis pneumonia (PcP). A number of studies show an association between exposure to
sulfonamides and an increased rate of point mutations in the dihydropteroate synthase (DHPS)
gene of P. jirovecii isolates. We are conducting studies in the Gauteng Province of South Africa,
with an estimated HIV prevalence of 14.5% and an incidence of PcP largely unknown. Our studies
aim to (1) estimate the burden of P. jirovecii infections in adults and children, (2) characterize the
local P. jirovecii strains, and (3) assess the prevalence of DHPS mutations in P. jirovecii isolated
from clinically defined patients. An initial pilot study, using Pneumocystis-positive specimens
received for routine laboratory diagnosis, was performed to determine the rate of DHPS mutations
in P. jirovecii strains from HIV-positive adults. PCR-amplified fragments of DHPS encompassing
the polymorphic positions were sequenced directly. Mutant alleles were detected in 38% of
specimens. A prospective clinical study in HIV-positive adult patients was subsequently launched
to investigate the interaction of sulfa prophylaxis, P. jirovecii DHPS mutations and clinical
outcome of PCP. Forty-four induced sputum specimens were obtained from 41 patients and
laboratory-confirmed Pneumocystis infection was found in 54% (22/41) of patients (17 females, 5
males). All cases were profoundly immunosuppressed; the median CD4 count (20/22 patients)
was 24 x 106/L (range 2--99 x 106) and none of the patients were receiving HAART. The
prevalence rate of DHPS mutations in the clinical study to date is 63.6%. The high prevalence of
P. jirovecii DHPS polymorphisms found in Gauteng Province contrasts with two previous studies
from the Western Cape Province that reported significantly lower rates of 1.9% and 13.3%,
respectively. Higher incidence of HIV infection in Gauteng Province and greater drug pressure
may be contributing factors to this geographical difference. We hypothesize that the general use of
sulfa drugs is exerting a selective pressure on P. jirovecii strains containing DHPS mutations in
South Africa.




                                                 146
P90C
Comparison of the substrate specificities of two Sap isoenzymes from Candida
parapsilosis
Olga Hruskova-Heidingsfeldova, Jiri Dostal, Martin Hradilek, Libuse Pavlickova and
Iva Pichova
Gilead Sciences Research Center, Institute of Organic Chemistry and Biochemistry,
Flemingovo n. 2, Prague 166 10, Czech Republic, Phone: +420 220183249, FAX: +420
220183578, e-mail: olga-hh@uochb.cas.cz, Web: www.uochb.cas.cz

Secreted aspartic proteinases of pathogenic Candida spp. represent one of the potential targets for
antifungal drug development. In our previous studies we have characterized substrate specificities
of purified Saps from C. albicans (Sap2p), C. tropicalis (Sapt1p) and C. parapsilosis (Sapp1p).
Candida parapsilosis, however, possesses three SAPP genes. We have purified two of the
respective proteins, Sapp1p and Sapp2p and compared their substrate specificities using sets of
synthetic substrates and inhibitors. Sapp1p can hydrolyze a wide range of peptides. Although it
prefers bulky hydrophobic residues between positions P3-P3´, it can cleave the peptides
containing polar amino acids, too. The specificity of Sapp2p is more restricted. Sapp2p strongly
prefers polar amino acid residues in either P1 or P1´ positions and does not hydrolyze bonds
formed by aromatic residues. The peptidomimetic inhibitors used in this study were either more
efficient in blocking Sapp1p, or comparable for both the isoenzymes tested. The only exception
was ritonavir, the clinically used inhibitor of HIV-1 proteinase. The Ki of ritonavir for Sapp2p was
one order of magnitude lower than for Sapp1p. This work was supported by the Czech Science
Foundation (grant No. 203/05/0038) and by Ministry of Education of the Czech Republic (grant
No. LC531).




                                                147
P91A
SNP analysis of antifungal resistance genes in human pathogenic Candida strains:
Bioinformatic solutions & prospects for ultra-deep sequencing
Bettina Rohde1, Sélène Ferrari2, Christopher Bauser1 and Johannes Regenbogen1
1
  , GATC Biotech, Jakob-Stadler Platz 7, Konstanz 78467, GERMANY, Phone: +49 (0)173
8160 0, FAX: +49 (0)173 8160 81, e-mail: c.bauser@gatc-biotech.com, Web: http://www.gatc-
biotech.com 2 Institut de Microbiologie, University Hospital Lausanne (CHUV), Swizerland

Mutations in the genes TAC1, PDR1 and ERG11 are responsible for efflux-based resistance
mechanisms to azoles. A collection of Candida strains resistant to this class of antifungals were
systematically classified. The DNA sequence of the complete coding region of the mentioned
genes was PCR amplified followed by high-throughput dideoxy Sanger sequencing and in silico
mutation analysis. More than 200 strains were analysed, generating 1500 overlapping PCR
fragments and 1500 sequencing reads. The resulting data were analysed using the
MutationSurveyor software (SoftGenetics) which provided a robust and accurate determination of
the base calls, base quality scores and mutation scores. This analysis method allowed the Euresfun
consortium to identify more than 100 mutations that are of potential relevance for the molecular
basis of azole resistance. The next-generation sequencing technologies now available will provide
an unparalleled opportunity to perform ultra-deep sequencing of these strains and recover
mutations that may only be present in a few percent of the cells in the isolate. We describe a state
of the art process of high throughput sequence data generation and processing as well as new
possibilities of ultra-deep comparative analysis of exons with clinical relevance. This work is
funded by the EC as part of the EURESFUN project FP6-518899.




                                                148
 P93C
Triclosan-mediated antagonism of the activity of azole antifungal drugs against Candida
albicans
Emmanuelle Pinjon, Gary Moran, Derek Sullivan and David Coleman
Department of Oral Biosciences, Dublin Dental Hospital, Lincoln Place, Dublin 2, Ireland,
Phone: +353 (0)1 612 7350, FAX: +353 (0)1 612 7295, e-
mail: emmanuelle.pinjon@dental.tcd.ie

Azole drugs are commonly used for the prophylaxis and treatment of candidosis in
immunocompromised individuals. However, drug interactions can affect the activity of these
drugs and could be associated with the development and/or expression of azole antifungal
resistance in Candida species. The aim of this study was to investigate whether resistance to
azoles can be induced in Candida species by exposure to triclosan, an antiseptic biocide with a
broad spectrum of activity against bacteria and yeasts, frequently incorporated to a wide range of
commonly used consumer products including toothpastes and mouthwashes. The bacterial target
of triclosan has been identified but little is known about its target in yeast species. Therefore, an
additional aim of this study was to investigate the mechanism(s) of action of triclosan in
Candida. The activity of triclosan in combination with azole drugs against C. albicans was
investigated. It was found that incubation of C. albicans with a subinhibitory concentration of
triclosan (1 g/ml) allowed significant increased growth in the presence of azole drug
concentrations that normally inhibit growth. This result suggested that triclosan can antagonise the
activity of azole drugs in vitro. Furthermore, in vitro sequential exposure of triclosan-susceptible
C. albicans and C. glabrata strains to increasing concentrations of triclosan resulted in the
recovery of derivatives exhibiting decreased susceptibility to fluconazole of 2-fold and 8–fold,
respectively. This result suggested that exposure to triclosan can induce resistance to fluconazole
in vitro. Transcriptional profiling was used to investigate the global expression of C. albicans
genes in response to triclosan exposure. The FAS1 and FAS2 genes, encoding the two subunits of
the fatty-acid synthase complex which acts as a functional homologue of the bacterial target of
triclosan, were found to be downregulated following 30 min exposure to triclosan. This suggested
that the mode of action of triclosan may involve this enzyme function in Candida as well. To
confirm this, we are in the process of constructing fas1 and fas2 knockout strains. The inactivation
of one allele of the FAS2 gene resulted in a 2-fold decrease in triclosan MIC. The fas2/FAS2 strain
also exhibited a marked reduction of the antagonism between triclosan and fluconazole. The
inactivation of the second FAS2 allele will be needed to confirm these preliminary results.




                                                149
P94A
Susceptibility to caspofungin and identification of FKS gene in Alternaria infectoria
Jorge Anjos, João Paulo Monteiro, António Meliço-Silvestre, Alexandra Abrunheiro and
Teresa Gonçalves
Centre for Neurosciences and Cell Biology, Institute of Microbiology, Faculty of Medicine,
Universidade de Coimbra, Rua Larga, Coimbra 3004-504, Portugal, Phone: +351-239834729,
FAX: +351-239826798, e-mail: jorge.f.anjos@gmail.com

Alternaria infectoria, a dematiaceous fungus, is a rare opportunistic agent of phaeohyphomycosis.
A case of cerebral alternariosis in a child, caused by A. infectoria, prompt us to study the
susceptibility of this fungus to caspofungin, and to identify putative homologues of the gene FKS,
that encodes for the catalytic unit of beta-1,3-glucan synthase, the caspofungin target. Besides our
fungal strain, several strains of A. infectoria isolated as aetiological agents of human infections,
were selected from the Central Bureau voor Schimmelculturen (CBS, Utrecht, The Netherlands)
fungal collection. The strains tested (with one exception) do not sporulate, so we developed and
optimised an inoculation protocol based on fragmented hyphae. The in vitro susceptibility to
caspofungin acetate (CAS) was studied with a microdilution assay [CLSI M38-A guidelines], and
by radial growth inhibition [1]. A concentration of 1 g/ml of caspofungin proved to inhibit the
radial growth of all the strains tested. In five of the eight strains, the microdilution assay showed
an average MEC value (defined as the minimal concentration able to induce significant
morphological changes) of 1.0 g /ml, with a range of 0.25 to 2 g /ml. These results were
obtained with an incubation period of 48 h, at 35ºC. The other strains did not exhibit visible
growth at 35 ºC, within a 72 h period but the MEC values were in the same range. We also report
the identification of a homologue of the fungal FKS gene in A. infectoria. The use of degenerated
oligonucleotides derived from the conserved regions of FKS genes of other fungi, allowed
identifying, a 5.5 kb sequence, AiFKS, which shares a sequence identity of 61% with Aspergillus
terreus and of 70% with Fusarium solani FKS genes. Taken together, the results obtained show
that A. infectoria has a gene homologue of the FKS genes of other fungi and that it exhibits
marked susceptibility to caspofungin in vitro. Nevertheless, we emphasize the difficulties
encountered to apply the conventional protocols to measure in vitro susceptibility to antifungals in
molds that do not sporulate and/or, as environmental ubiquitous species, hardly grow at 35 ºC, the
standard temperature used to measure antifungal break points.

[1] Kahn, J.N. et al., Antimicrob. Agents Chemother., 50, 2214.

This work was supported by a research Medical School Grant from Merck & Company.




                                                150
P95B
Antifungal target discovery and validation
Michael Bromley, Paul Carr, Sandra Howsley, Sarah Kaye, Danny Tuckwell and Jason Oliver
Targets, F2G Ltd, PO Box 1, Lankro Way, Eccles, Manchester M30 0BH, UK, Phone: +44 (0)
161 275 1270, FAX: +44 (0) 161 785 1273, e-mail: jasonoliver@f2g.com,
Web: http://www.f2g.com/

With the aim of discovering new antifungal drugs, F2G employs both traditional whole-cell
screening and target-based screening. Our target discovery technology platform, Mycobank®,
identifies genes that are essential for the growth of Aspergillus fumigatus. Various target
validation processes, employing both bioinformatics and experimental approaches, determine
whether these genes will make good antifungal drug targets. Spectrum, selectivity, essentiality
and function are considered. Importantly, the target must be amenable to high-throughput
screening. Selected genes are then chosen for assay and screen development. Usually this
requires a source of protein; so recombinant target protein is prepared, either in heterologous or
homologous expression systems. A variety of target-based screens have been developed at F2G
employing different assay types to look at novel targets.




                                                151
P96C
Detection and quantification of gene expression associated with resistance to azoles in
clinical strains of Candida albicans
Elisabete Ricardo1, Sofia Costa-de-Oliveira1, Ana Silva-Dias1, Acácio Gonçalves-Rodrigues2
and Cidália Pina-Vaz3
1
  Microbiology, Faculty of Medicine Porto University, Alameda Prof. Hernani Monteiro, Porto
4200-319, Portugal, Phone: +351 225513662, FAX: +351 225513662, e-
mail: betaricardo@yahoo.com 2 Department of Microbiology, Faculty of Medicine, University
of Porto, Porto Portugal; Burn Unit, Department of Plastic and Reconstructive Surgery, Faculty
of Medicine, University of Porto and Hospital S. João, Porto, Portugal 3 Department of
Microbiology, Faculty of Medicine, University of Porto, Porto Portugal; Department of
Microbiology, Hospital S. João, Porto, Portugal

Emerging antifungal resistance among Candida spp is a major problem especially for
immunosuppressed patients. Azoles are widely used for therapy, cross-resistance being frequent.
Overexpression of efflux pumps encoded by CDR1, CDR2 and MDR1 genes and/or an alteration
of the target protein, encoded by ERG11 are the most studied resistance molecular mechanisms.
Previously, we have quantified CDRs gene expression in resistant (R) clinical strains of C.
albicans comparing to susceptible (S) ones. Some of those R strains did not present a high CDR
gene expression, other mechanisms being involved. The aim of this work was to undertake a more
detailed gene expression study, in R clinical strains of C. albicans, involving also MDR1
(belonging to major facilitator family of proteins and mostly associated to resistance to
fluconazole) and ERG11 (encoding one of the azole target enzymes conferring resistance either by
point mutations and/or increased levels of its expression), establishing a relationship between gene
expression and azole phenotype. Forty clinical (20 R and 20 S) and 3 control strains (with known
resistance mechanisms; kindly gifted by Dr Theodore White) of C. albicans were studied. After
incubation overnight at 37ºC, they were harvested on exponential growing phase (OD600=0.5)
and immediately frozen at -70ºC. Total RNA extraction was performed using phenol-chloroform
method, the RNA quality evaluated by electrophoresis and its absorbance determined at
260/280nm. A Reverse Transcriptase PCR reaction was performed and cDNA amplified in a two
step Real Time PCR (LightCycler from Roche). SYBR Green (QIAGEN) was used for
quantification and melting curves introduced at the end of each amplification cycle to assure
specificity. Specific primers were designed for MDR1 and ERG11 genes; ACT1 was used as
normalizing gene. Most of azole cross-resistant strains displayed an increased expression of CDR
and in MDR genes; however 4 strains (R to fluconazole) showed only a significant increase in
MDR expression. Two strains showed an increase of ERG11 expression. The differences were
statistically significant between R and S strains (p<0.05). A detailed study involving the
expression of resistance genes was performed in clinical strains of C. albicans in order to better
clarify the molecular mechanism of resistance. The knowledge of such mechanisms could allow
its reversion, with considerable impact upon patient outcome.




                                                152
P98B
Study on the localization of Gas1, Gas2 and Gas4 proteins during vegetative growth and
during sporulation in yeast
Julia Calderon, Eleonora Rolli and Laura Popolo
Dipartimento di Scienze Biomolecolare e Biotecnologie, Università di Milano, Via Celoria 26,
Milano It 20133, Italy, Phone: +39(0)25031 4920, FAX: +39(0)25031 4912, e-
mail: julia.calderon@unimi.it, Web: www.unimi.it

Cell walls are dynamic structures that are essential for almost every aspect of yeast and fungi
biology, including viability, morphogenesis and pathogenesis. Transglucosidases play a significant
role in the biosynthesis of the cell wall, as they are required for correct assembly of its
components. Members of the Family 72 of Glycoside Hydrolases (GH) are endowed with
beta(1,3)-Glucanosyltransferase activity. These proteins are either anchored to the plasma
membrane through a GPI or cross-linked to the cell wall. In S. cerevisiae five paralogous genes,
GAS1-5, encoding members of the Family GH72 have been identified. GAS1 and GAS5 genes are
expressed during vegetative growth and repressed during sporulation whereas GAS2 and GAS4
have the reverse expression pattern. Besides GAS3 is weakly expressed both during vegetative
growth and sporulation. We studied the localization of Gas1p during vegetative growth and of
Gas2 and Gas4 proteins during sporulation. To carry out these analysis we used techniques of
direct visualization of Fluorescent-Fusion proteins and indirect Immunofluorescence. In order to
examine the localization of Gas1p, an N-terminal tagging of an improved version of the Red
Fluorescent Protein (mRFP) was perfomed using the MF608 plasmid (1). Through an integrative
transformation we introduced the Gas1-mRFP cassette in the WAH strain (gas1-delta) derived
from W303-1A. Two recombinant clones were found to revert the hypersensivity to Calcofluor
White of gas1 null cells. Fluorescent microscopic analysis showed a localization on the cell
surface surface and at a higher level at the bud neck By indirect immunofluorescence technique
we also identified a Gas2-3X HA protein version using anti-HA monoclonal antibody and Gas4p
using polyclonal antibodies raised against the recombinant protein. Both Gas2 and Gas4 proteins
were detected at the surface of ascopores at 24 hours after the induction of sporulation consistently
with their role in spore wall maturation (2).

This work was supported by the CanTrain EU project (MRTN-CT-2004-512481) and by COFIN
2005 to L. P.

References:
1. Fujita M., et al. (2006). Inositol deacylation by Bst1p is required for the quality control of
glycosylphosphatidylinositol-anchored proteins. Mol Cell Biol. 17(2): 834-50.
2. Ragni E., et al. (2007). GAS2 and GAS4, a pair of developmentally regulated genes required for
spore wall assembly in Saccharomyces cerevisiae. Eukaryot. Cell. 6: 302-316.




                                                153
P99C
Novel mutations in a Saccharomyces cerevisiae ABC protein, Pdr5p, result in insensitivity
to the efflux pump inhibitor FK506
Koichi Tanabe1, Susumu Tomiyama1, Erwin Lamping2, Yukie Takano1, Yoshimasa Uehara1
and Masakazu Niimi1
1
  Deaprtment of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1
Toyama, Shinjuku-ku, Tokyo 162-8640, JAPAN, Phone: +81 (0)3 5285 1111, FAX: +81 (0)3
5285 1272, e-mail: niimi@nih.go.jp 2 Department of Oral Sciences, University of Otago,
Dunedin, New Zealand

Most ATP binding cassette (ABC) transporters are membrane proteins whose over-expression
frequently confers resistance to antifungal drugs including the widely used and well-tolerated
azole antifungal fluconazole. FK506, a commonly used immunosuppressant drug, is known to
inhibit some fungal ABC transporters, but its interaction with the transporters is incompletely
elucidated. This study describes the inhibitory effect that FK506 has on the fungal multi-drug
resistant transporter Pdr5p from Saccharomyces cerevisiae. The inhibitory effect of FK506 makes
fluconazole resistant Pdr5p over-expressing S. cerevisiae cells highly sensitive to fluconazole.
Thirty-nine FK506-insensitive mutants of Pdr5p over-expressing strains were isolated on agar
plates that contained sub-MIC concentration of fluconazole together with FK506. This screen was
designed to isolate FK506 resistant mutants of Pdr5p that are otherwise fully functional and able
to transport fluconazole. Sequence analysis revealed that each mutant had a point mutation in the
PDR5 ORF. The mutations were mapped and mostly located in the predicted external loops or
outer regions of the transmembrane helices of the Pdr5p molecule. The mutant strains were
resistant to FK506 but fully maintained their pumping activity with MICs of fluconazole and other
pump substrates similar to the parental Pdr5p over-expressing strain. The inhibitory effect of
FK506 on the mutant Pdr5p activity was also evaluated by measuring its inhibitory effect on the
transport of rhodamine 6G, a fluorescent pump substrate, and the in vitro pump ATPase activity.
Mutations of Pdr5p detected in this study eliminated the inhibitory effect of FK506 on Pdr5p
without affecting its drug transport activities. These results suggest that FK506 directly interacts
with Pdr5p, being potentially recognized as a substrate that has a high affinity to the Pdr5p pump
in its open (substrate release) conformation and therefore locks the pump in its open conformation
thus inhibiting transport of other substrates.




                                                154
P100A
Ergosterol biosynthesis characterization in Aspergillus fumigatus by sterol analysis
Laura Alcazar Fuoli1, Emilia Mellado Terrado1, Guillermo Garcia Effron1, Jordi F. Lopez2,
Joan O'Grimalt2, Manuel Cuenca Estrella1 and Juan Luis Rodriguez Tudela1
1
  Servicio Micología, Centro Nacional Microbiología, CNM, Ctr. Pozuelo-Majadahonda Km 2,
Madrid sp 28220, spain, Phone: +34918223661, FAX: +34915097034, e-
mail: lalcazar@isciii.es, Web: www.isciii.es 2 Departamento de Química Ambiental. Instituto
de Investigaciones Químicas y Ambientales de Barcelona “Josep Pascual Vila” (IIQAB).
Consejo Superior de Investigaciones Científicas. 08034 Barcelona (Spain).

Background: The Aspergillus fumigatus ergosterol biosynthesis is of interest since this pathway
is the target for many antifungal drugs in clinical use. The conversion from squalene to ergosterol
is a complex pathway in which about 20 enzymes are involved. Since very little is known about
ergosterol biosynthesis in A. fumigatus, the sterol intermediates leading to ergosterol were
analyzed in different strains. A. fumigatus strains included azole susceptible (AZL-S) and AZL-
resistant (AZL-R) strains and also, A. fumigatus mutants deficient in different enzymatic steps,
such as 14-&#945;-sterol demethylases (Cyp51A and Cyp51B) and C-5 sterol desaturases (Erg3A,
Erg3B and Erg3C) were analysed.

Methods: The CM-237, strain (AZL-S) was used as the reference strain. AZL-R strains (CM-
2159, CM-3269, CM-0796) and mutant strains: CM-A8 (cyp51A-), CM-B7 (cyp51B-), CM-A80
(erg3A-), CM-B866 (erg3B-), CM-C65 (erg3C-). Sterols were analyzed by GC-MS and
identification was based on mass spectral interpretation, comparison of mass spectra and retention
time data with available standards.

Results: Fourteen different sterols where identified in the A. fumigatus wild type strain: (1) 24-
methylcholesta-5,7,9(11),22-tetraen-3ß-ol, (2) 24-methylcholesta-5,8,22-trien-3ß-ol, (3) 24-
methylcholesta-5,7,9,22-tetraen-3ß-ol, (4) 24-methylcholesta-5,7,22-trien-3ß-ol, (5) 24-
methylcholesta-7,22-dien-3ß-ol, (6) 24-methylcholesta-5,7,22,24(28)-tetraen-3ß-ol, (7) 24-
methylcholesta-7,22,24(28)-trien-3ß-ol, (8) 24-methylcholesta-5,7,24(28)-trien-3ß-ol, (9) 24-
methylcholesta-7,24(28)-dien-3ß-ol, (10) 24-Ethylcholest-5,7,22-trien-3ß-ol, (11) 4,4,14-
trimethylcholesta-8,24-dien-3ß-ol,     (12)     4,24-dimethylcholesta-8,24(28)-dien-3ß-ol,    (13)
4,4,14,24-tetramethylcholesta-8,24(28)-dien-3ß-ol, (14) 4,4,24-trimethylcholesta-8,24(28)-dien-
3ß-ol. Analysis of the sterols derivatives in A. fumigatus enzyme defective strains allowed the
description of the ergosterol biosynthesis pathway in this important fungi.

Conclusions: (i) A description of the ergosterol biosynthesis pathway in A. fumigatus is initially
attempted. (ii) Sterols in A. fumigatus AZL-R strains were qualitatively and quantitatively similar
to those in the AZL-S strain. (iii) Lanosterol is methylated in C-24 to give a sterol of 31 carbons
on which the 14-&#61537; sterol demethylase would act and after its product would be
demethylated at C-4. (iv) In A. fumigatus the steps from fecosterol to ergosterol may proceed
through various routes to give ergosterol as the final product.




                                               155
P101B
Identification of genes involved in biofilm formation in Candida glabrata
Marta Riera1, Estelle Mogensen1, Sophie Goyard2, Christophe d'Enfert2 and Guilhem Janbon1
1
  Mycologie Moléculaire, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France,
Phone: +33 (0)1 40 61 83 56, FAX: +33 (0)1 45 68 84 20, e-
mail: estelle.mogensen@pasteur.fr, Web: http://www.pasteur.fr/ 2 Biologie et Pathogénicité
fongiques, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France

Medically implanted devices provide surfaces on which human pathogenic yeast such as
Candida albicans and Candida glabrata can adhere and develop as biofilm structures.
Biofilms have been shown to be more resistant to antimicrobial agents and host defence
machinery than planktonic cells, making infections more difficult to treat. However, little is
known about specific genes involved in the formation and the growth of biofilms in fungi. A
genetic screen for Candida glabrata mutants impaired in biofilm formation was developed
using a 96-well plate biofilm model. Several genes were identified and the corresponding
proteins isolated, including Epa6p which is an adhesin essential for biofilm formation and the
protein kinase Yak1p which is required for the expression of EPA6 . Yak1p acts through a
subtelomeric silencing machinery-dependent pathway which requires other proteins such as
Sir3p, Sir4p and Rif1p (Iraqui et al. , 2004). It has been demonstrated that Yak1p is also
necessary for biofilm formation in Candida albicans, which validates the Candida glabrata
model for the identification of genes involved in biofilm formation in pathogenic yeasts. In
order to identify new elements involved in the Yak1p pathway that controls biofilm formation,
additional biofilm mutants have been characterised using the same genetic screen. This way,
the complex Swi/Snf and the transcription factor Cst6p have been showed to play an important
role in biofilm development. Currently, genetic and biochemical approaches are being used in
order to determine how these different components interact with each other.

Iraqui, I, et al., (2004), Mol. Microbiol., 55, 1259




                                                  156
P102C
Substrate specificity and function of Candida albicans general amino-acid permeases
(CaGaps)
Lucie Kraidlova1, Mycola Maidan2, Patrick van Dijck2 and Hana Sychrova1
1
  Department of Membrane Transport, Institut of Physiology, Videnska 1083, Prague 14220,
Czech Republic, Phone: +420 24106 1111, FAX: +420 24106 2488, e-
mail: kraidlova@biomed.cas.cz 2 Department of Molecular Microbiology, Institute of Botany
and Microbiology, K.U. Leuven, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium

Candida cells can proliferate in many different niches within the host and must be able to sense
the environment in order to express only those genes that help to utilize optimally all nutrient
sources in the area. Sensing and uptake of amino acids, which are present in mammalian hosts
in high concentration and which constitute major source of nitrogen, would have a central role
in the growth of C. albicans. Also the requirement of amino-acid transporters for hyphal
morphogenesis induction and virulence was shown. In Saccharomyces cerevisiae, one general
amino-acid permease (Gap1p) exisits. It is not only required for transport, but also for amino-
acid sensing and activation of signal transduction pathways that induce many intracellular
changes. In C. albicans, a whole family (5 members) of Gap1 homolog exists. We would like
to elucidate the role of individual CaGap permeases in the virulence and pathogenicity of this
species, together with the characterization of their transport properties. For this, we employ
deletion of one or both GAP alleles in the C. albicans SC5314 strain (using Ura-blaster
strategy; Fonzi, W.,et al., (1993), Genetics, 134, 717), and/or putting one allele under a
regulatable promoter, followed by a detailed physiological characterization of C. albicans
mutants, together with the heterologous expression of CaGAP genes in a S. cerevisiae mutants
lacking their own amino-acid transporters (can1 lyp1 gap1) to reveal the substrate specificity
and kinetic parameters of individual CaGap permeases.
This work was supported by the Czech grants GA CR 204/03/H066, MSMT LC 531, and by
the Czech-Flemish bilateral project 1-2006-06.




                                               157
P103A
Peroxisomal compartmentalization of the glyoxylate cycle in Candida albicans: evidence
for strongly reduced glyoxylate cycle function in giant peroxisomes
Katarzyna Piekarska2, Guy Hardy1, Carlo van Roermund3, Karin Strijbis1, Els Mol1, Janny van
den Burg1, Marlene van den Berg1 and Ben Distel1
1
  Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15,
Amsterdam 1105 AZ, The Netherlands, Phone: +31-(0)20 5665127, FAX: +31-(0)20 6915519,
e-mail: b.distel@amc.uva.nl 2 University of Manchester, The Michael Smith Building, Oxford
Road, Manchester M13 9PT, UK 3 Department of Genetic Metabolic Diseases, Academic
Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

The glyoxylate cycle, a metabolic pathway required for generating C4 units from C2
compounds, is an essential factor in virulence, both in animal and plant pathogens. Here, we
report the localization of the key enzymes of this cycle, isocitrate lyase (ICL) and malate
synthase (MLS), in the human fungal pathogen Candida albicans. Immunocytochemistry in
combination with subcellular fractionation showed that both ICL and MLS are localized in
peroxisomes independent of the carbon source used. Although ICL and MLS lack a consensus
type I peroxisomal targeting signal (PTS1), their import into peroxisomes was dependent on
the PTS1-receptor Pex5p, suggesting the presence of non-canonical targeting signals in both
proteins. Peroxisomal compartmentalization of the glyoxylate cycle is, however, not essential
for proper functioning of this metabolic pathway because a pex5 knock out strain, in which
ICL and MLS were localized to the cytosol, grew equally well as the wild type strain on
acetate and ethanol. Previously, we reported that a fox2 deletion strain that is completely
deficient in fatty-acid beta-oxidation but has no peroxisomal protein import defect, showed a
strongly reduced growth on acetate and ethanol [1]. Here, we present evidence that the fox2
deletion strain is disturbed in the transport of glyoxylate cycle products and/or acetyl-CoA
across the peroxisomal membrane and discuss the possible relationship between such a
transport defect and the presence of giant peroxisomes in the fox2 mutant.

1. Piekarska, K. et al., (2006), Euk. Cell, 5, 1847




                                                 158
P104B
Biofilm formation ability by non-Candida albicans Candida species
Sónia Silva1, Mariana Henriques1, Rosário Oliveira1, David Williams2 and Joana Azeredo1
1
  Biological Engineering, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal,
Phone: +351 253604409, FAX: + 351 253678986, e-mail: soniasilva@deb.uminho.pt 2 School
of Denstistry, Cardiff University, United Kingdom

The number of infections caused by Candida species has greatly increased in the past ten
years. This has been attributed to associated increases in the number of AIDS patients, the
increasingly elderly population and the number of immunocompromised patients. Moreover,
the increased use of indwelling medical devices has also been implicated with the rise of
candidal infections. Most candidoses have been attributed to Candida albicans, however,
recently, new non-Candida albicans Candida (NCAC) species have been identified as common
pathogens, namely C. parapsilosis, C. glabrata, C. tropicalis, C. krusei and C.
dubliniensis. Biofilms are the most frequent form of environmental microbial growth and play
an important role in clinical infections. Of significance is that biofilms tend to resist removal
by host factors and administered antimicrobials and therefore represent a persistent source of
infectious organisms. The aim of this study was to assess the ability of NCAC species to
produce biofilms. A total of 16 NCAC strains isolated from the vagina, urinary and oral tract
were used, including C. parapsilosis (n=6), C. glabrata (n=6) and C. tropicalis (n=4).
Reference strains of each species (C. glabrata IGG 2418, C. tropicalis IGC 3097T/CBS94 and
C. parapsilosis 37) were similarly examined. Biofilms were formed in 96-well microtitre
plates, in Sabouraud dextrose broth at 37ºC (agitated at 130 rpm). The ability of biofilm
formation was assessed after 48h through total biomass quantification by crystal violet staining
and cellular activity by the reduction of a tetrazolium salt (XTT). The results showed that most
NCAC species were able to form biofilms, although there were differences depending on
species, strain or isolate origin. Comparison of biofilm biomass with cell activity did not reveal
any correlation, probably due to different amounts of extracellular matrix produced by each
strain.




                                                159
P105C
PIR1 polymorphism as a tool for Candida albicans identification in Candida infections
Inmaculada Moreno1, Ana Isabel Martinez2, Luis Castillo1, Lucas del Castillo1,
Rafael Sentandreu1 and Eulogio Valentin1
1
  Microbiology and Ecology, Faculty of Pharmacy. University of Valencia, Avda. Vicente
Andres Estelles s/n, Burjassot- Valencia 46100, Spain, Phone: +34 963544684, FAX: +34
963544543, e-mail: inmaculada.moreno@uv.es 2 Molecular Recognition Laboratory. Príncipe
Felipe Research Centre, Valencia, Spain.

Pir proteins (Protein with internal repeats) were firstly described in Saccharomyces cerevisiae.
The S. cerevisiae Pir family consists of four members which present some characteristic
features: (i) these proteins are synthesized as pre-proteins, and processed by the serine
proteinase Kex2 in the Golgi, (ii) contain a variable number of internal repeats matching the
consensus pattern Q[IV]XDGQ[IVP]Q, and (iii) have a conserved C-terminal domain
containing four cysteine residues. It has been suggested that the role of the internal repeats of
Pir proteins is important for cell wall anchoring to beta-1,3-glucan (Castillo et al., 2003). In the
C. albicans parental strain SC5314, only one Pir protein has been identified. The two alleles of
C. albicans PIR1 gene encode for two different-size cell wall mannoproteins with 9 and 7
internal repeats (-Q-I-(S/T/G/N)-D-G-Q-(I/V)-Q-H-Q-T-) respectively in their aminoacid
sequences. Heterozygous mutants in each of the two alleles of PIR1 gene present abnormal
phenotypes; since the homozygous mutant has not been obtained yet, we suggest that PIR1
plays an essential role in C. albicans (Martinez et al., 2004). A search for PIR genes in a
collection of several Candida strains (albicans and non-albicans) was done by PCR using a
pair of primers that amplify the CaPIR1 ORF. This study showed that the pair of
oligonucleotides designed as primers was specific for C. albicans, as no amplification was
obtained when DNA from non-albicans species were used as template. Moreover, a collection
of 43 C. albicans clinical isolates from different sources was also tested. The PCR products
obtained for these samples revealed that other PIR1 patterns, different from the SC5314, exist.
Six different patterns were obtained among the isolates tested as combination of the four PIR1
length variants found, only differing in the amount of internal repeats. Further studies should
help us to elucidate whether there is any correlation between the amount of internal repeats in
Pir1 proteins and the clinical importance of different strains.

REFERENCES:
Castillo, L, et al., (2003). Yeast 20:973.
Martínez, A.I., et al., (2004). Microbiology 150:3151.




                                                 160
P106A
Defence of Aspergillus fumigatus against reactive oxygen species mediated by Afyap1
Franziska Lessing, Olaf Kniemeyer and Axel A. Brakhage
molecular and applied microbiology, Leibniz Inst. for natural prod. research + infection
biology, Beutenberg str. 11a, Jena 07745, Germany, Phone: +49 (0) 3641 656817, FAX: +49
(0) 3641 656603, e-mail: franziska.lessing@hki-jena.de

With the increasing number of immunocompromised individuals Aspergillus fumigatus has
become one of the most important opportunistic fungal pathogens. During infection A.
fumigatus is confronted with a number of defence mechanisms in the host, particulary
neutrophiles and macrophages, which kill conidia by producing reactive oxygen species. We
identified a homolog of the AP1 like transcription factor Yap1 from yeast in A. fumigatus,
which we designated Afyap1. In yeast, Yap1p was found to be a global regulator for oxidative
stress response and required for the protection of the cell against H2O2 and other reactive
oxygen species (ROS). Similarly, deletion of Afyap1 led to A. fumigatus mutant strain which
showed drastically increased sensitivity against ROS. Nuclear localisation of an Afyap1-eGFP
fusion in A. fumigatus was dependent on the presence of H2O2 and diamide. To identify new
targets of Afyap1, we compared the proteome pattern of H2O2 treated and non-treated wild-
type mycelia and of a Afyap1 deletion strain by 2D-gel analysis. Moreover, despite the
importance of Afyap1 for defence against ROS, the Afyap1 deletion mutant was not reduced in
pathogenicity in a low dose murine infection model for invasive aspergillosis. These data
indicate that at least in the mouse infection model, ROS do not play a significant role in killing
of A. fumigatus by immune effector cells.




                                                161
P107B
Proteome analysis of the response of Aspergillus fumigatus to iron limitation
André D. Schmidt1, Olaf Kniemeyer1, Hubertus Haas2 and Axel A. Brakhage1
1
  Molecular and Applied Microbiology, Hans-Knoell-Institute / Friedrich-Schiller-University
Jena, Beutenbergstr. 11a, Jena 07745, Germany, Phone: +49 3641 656858, FAX: +49 3641
656603, e-mail: andre.schmidt@hki-jena.de, Web: www.hki-jena.de 2 Division of Molecular
Biology, Biocenter, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck,
Austria

The acquisition of iron is known to be an essential step in any microbial infection process due
to iron-limiting conditions in the human host. This iron limitation is caused by high-affinity
iron-binding proteins like transferrin or lactoferrin in the host. Since iron plays an essential role
in key metabolic processes like DNA synthesis, oxidative phosphorylation or electron transport
A. fumigatus has to overcome the iron deficiency by the synthesis of siderophores, which
chelate iron. It was shown that an A. fumigatus strain unable to synthesize siderophores was
attenuated in virulence in a murine infection model. To understand the cellular processes,
induced by iron starvation, we analysed the proteome of A. fumigatus strain ATCC 46645
grown under iron-deficiency conditions. Under iron depletion, proteins involved in siderophore
biosynthesis are upregulated, e.g. L-ornithine N5-oxygenase (SidA), and iron cluster-containing
proteins as aconitase or 3-isopropylmalate dehydratase are down-regulated. In addition,
proteins involved in the heme biosynthesis are less abundant under iron-deficiency. Further
proteins analysed under different non-linear pH-scales will be presented and their putative role
will be discussed.




                                                  162
P108C
Targeted gene deletion of Candida parapsilosis lipase genes CpLIP1 and CpLIP2
Attila Gacser1, David Trofa1, Joachim Morschhäuser2, Wilhelm Schäfer3 and Joshua
D. Nosanchuk4
1
  Department of Medicine, Infectious Diseases, Albert Einstein College of Medicine
(AECOM), 1300 Morris Park Ave, New York NY 10461, USA, Phone: +1 718 430 2993,
FAX: +1 718 430 8968, e-mail: gacsera@gmail.com 2 Institute for Molecular
Infectionsbiology, University of Würzburg, Röntgenring 11, 97070 Würzburg, Germany 3
Department of Molecular Phytopathology and Genetics, Biocenter Klein Flottbek, University
of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany 4 Department of Medicine,
Department of Microbiology and Immunology AECOM

Candida parapsilosis is currently the second most common cause of invasive candidiasis. It is
particularly associated with disease in premature infants and immunocompromised adults, and
as a nosocomial infection in Intensive Care Units. Although it is assumed that secreted lipases
play an important role in microbial virulence, the involvement of these enzymes in C.
parapsilosis virulence has not been precisely defined. We successfully adapted a method for
sequential gene disruption in C. albicans that is based on the repeated use of the dominant
nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-
specific recombination (Reuss O et al 2004, Gene, 341, 119) for use in C. parapsilosis. We
designed a knock out construct to target the lipase locus in the C. parapsilosis genome
consisting of adjacent genes, CpLIP1 and CpLIP2. Our results showed that this locus had two
copies in the genome. The close genomic localization enabled the deletion of the CpLIP1 and
CpLIP2 genes using one knock-out vector to generate a homozygous “lipase minus” strain.
After the gene deletion we reconstructed the CpLIP2 gene, which restored lipase activity.
Lipolytic activity was absent in the null mutants, whereas the wild type, heterozygous and
reconstructed mutants showed similar lipase production. In YPD and minimal YNB medium
there were no significant differences in the growth of the wild type and mutant strains. In
contrast, in comparison to wild-type C. parapsilosis the growth of lipase minus mutants was
significantly reduced in both YNB medium with olive oil (3 fold reduction) and YNB
supplemented with intralipid parenteral nutrition (1.5 fold reduction, p=0.02) suggesting that
lipases are important for providing the fungus with carbon and energy sources. Biofilm
formation, which is important for candidal virulence, was reduced in the lipase deficient strain
that had a 5 fold reduction (p=0.008) in XTT activity compared to controls after 48h growth on
polystyrene surfaces. These studies represent the first demonstration of targeted gene
disruption in C. parapsilosis. Additionally, we show that C. parapsilosis lipases facilitate
biofilm formation and fungal growth in lipid-rich environments. Future studies with the lipase
minus strains will investigate their fitness in different murine infections models.




                                               163
P109A
Development of an in vivo subcutaneous Candida albicans biofilm model
Marketa Ricicova1, Katrien Lagrou2 and Patrick Van Dijck1
1
  Department of Molecular Microbiology, VIB, Katholieke Universiteit Leuven, Kasteelpark
Arenberg 31, Leuven-Heverlee 3001, Belgium, Phone: +32 (0)16 32 1500, FAX: +32 (0)16 32
1979, e-mail: Marketa.Ricicova@bio.kuleuven.be, Web: http://bio.kuleuven.be/mcb 2
Department of Medical Diagnostic Sciences, Katholieke Universiteit Leuven

The majority of microbes in their natural habitats are not found as free-living organisms but
rather in structured communities attached to surfaces, commonly referred to as biofilms.
Biofilm formation caused by opportunistic fungal pathogen C. albicans has significant clinical
consequences, as adhesion to inert surfaces such as urinary or central venous catheters, dental
prostheses, and other biomaterials can lead to a failure of these implanted devices. In addition,
C. albicans biofilms are often extremely resistant to drug therapy and can act as a source of re-
infections. Most of the Candida biofilm studies have so far involved in vitro models where the
host immune system is not considered. Though, recently there have been published two in vivo
central venous catheter models in rats and rabbits (1 and 2). However, these systems require
rather difficult procedures which are not convenient for high throughput studies. Therefore, we
try to develop an easier in vivo model with catheters implanted under the skin of rats based on
a successful bacterial subcutaneous model (3). Catheters carrying biofilms are explanted after
several days and are subjected to fluorescence and scanning electron microscopy. The amount
of cells present in biofilm is determined by colony forming units and quantitative PCR. Once
the optimal conditions are settled, we will test different C. albicans mutant strains in this
model and compare it to in vitro test results. Gene expression study of the biofilm growth in
both systems will be performed as well. The new in vivo Candida biofilm model could easily
serve as a tool for investigation of novel drug therapies.

References:
1. Andes D. et al.(2004), Infect Immun., 72(10),6023
2. Schinabeck M. et al., (2004), Antimicrob Agents Chemother., 48(5),1727
3. Van Wijngaerden E. et al.(1999), J Antimicrob Chemother., 44(5),669




                                                164
P110B
Effect of oral pathogens on Candida biofilms on different denture materials
Tatiana Pereira1, Ephie Kraneveld1, Altair Del Bel Cury2, Erik Manders3, Dongmei Deng1,
Bob ten Cate1 and Wim Crielaard1
1
  Cariology Endontology Pedodontology, Academic Centre for Dentistry Amsterdam,
Louwesweg 1, Amsterdam 1066 EA, The Netherlands, Phone: +31 20 5188596, FAX: +31 20
6692881, e-mail: T.Pereira@acta.nl, Web: www.acta.nl 2 Piracicaba Dental School, State
University of Campinas, Brazil 3 Swammerdam Institute of Life Sciences, University of
Amsterdam

Although different materials and liners are being used in dental practice, denture stomatitis,
induced by Candida species together with oral bacteria is still a major problem. To understand
the complex interactions that exist between oral surfaces, dietairy sugars, saliva, eukaryotic and
prokaryotic micro-organisms and Candida infections we evaluated the effect of oral pathogenic
bacteria (e.g. Lactobacillus plantarum) on in vitro Candida albicans and Candida glabrata
biofilm development under different environmental conditions. Biofilms of C. albicans ATCC
90028 and C. glabrata ATCC 90030 were grown as single and multi-species biofilms on
hydroxyapatite, denture base and denture relining material discs in an artificial saliva medium
supplemented with glucose or sucrose. Biofilms were grown for 24h and analyzed every two
hours for the number of viable cells (CFU), cell morphology (light micropscopy) and biofilm
structure (confocal laser microscopy). Results showed a statistical difference on multi-species
biofilm development when compared to single species, which could be modulated by
variations in the medium, but most notably by the type of denture material used. Confocal laser
microscopy and light microscopy showed differences in biofilm structure and cell morphology
for C. albicans and biofilm structure of C. glabrata as a result of the presence of oral
pathogens, saliva or medium changes. Specfically the effect of saliva is an important parameter
in evaluating the applicability of different denture materials.




                                                165
P111C
Secreted aspartic proteinases are not required for invasion of reconstituted human
epithelia by Candida albicans
Ulrich Lermann and Joachim Morschhäuser
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg
97070, Germany, Phone: +49 (0) 931 312127, FAX: +49 (0) 931 312578, e-
mail: u.lermann@mail.uni-wuerzburg.de

The yeast Candida albicans is a harmless commensal on mucosal surfaces in many healthy
people, but it can also cause superficial as well as life-threatening systemic infections,
especially in immunocompromised patients. Secreted aspartic proteinases (Saps) contribute to
virulence of C. albicans by facilitating invasion into host tissue, destroying host defense
molecules, and providing nutrients for growth. The Saps are encoded by a multigene family of
ten members which are differentially expressed within the host, depending on the site and stage
of an infection. The role of individual Sap isoenzymes has previously been investigated by
inactivating the corresponding genes in an auxotrophic C. albicans laboratory strain. However,
it has recently become evident that this approach may lead to wrong conclusions about the
importance of target genes in virulence due to inappropriate expression of the URA3 nutritional
marker used for mutant construction. By applying a novel method that allows gene disruption
in C. albicans wild-type strains with the help of a recyclable dominant selection marker, we
constructed mutants of C. albicans strain SC5314 lacking individual (sap1, sap2, sap3, sap4,
sap5, sap6) or multiple (sap123 and sap456) SAP genes and tested their ability to invade and
destroy reconstituted human epithelium (RHE). In contrast to a non-filamentous efg1 mutant,
which was unable to invade into the epithelium, all sap mutants were as invasive as the wild-
type parental strain. Therefore, we conclude that the secreted aspartic proteinases are not
required for epithelial invasion and tissue damage in these in vitro models of candidiasis.




                                               166
P112A
Combining post genomics and infection models to elucidate Candida albicans-host
interactions during the disease process
Duncan Wilson and Bernhard Hube
Microbial Pathogenicity Mechanisms, Hans Knöll Institut, Beutenbergstrasse, Jena D-07745,
GERMANY, Phone: +49 (0) 3641 65 68 85, FAX: +49 (0) 3641 65 68 82, e-
mail: Duncan.Wilson@hki-jena.de, Web: http://www2.hki-jena.de/rz/hki_i00.htm

Transcriptional profiling of C. albicans in response to specific host environments is a useful
tool for determining the mechanisms this pathogen employs to establish and maintain
infection. Full appreciation of such data sets however is frequently hampered by the large
proportion of unknown function genes which are differentially regulated. To understand the
behaviour of C. albicans in vivo, we have performed transcriptional profiling of the organism
during a number of in vivo, in vitro and ex vivo infection models. Comparative analysis has
identified one gene of unknown function which is commonly upregulated in response to: blood
components, invasion of intraperitoneal organs and an in vitro model of oral infection. in silico
analysis of the predicted amino acid sequence suggests that this gene encodes a membrane
protein. In addition, a number of genes of unknown function upregulated specifically by the
host microenvironment have been identified and analysed in silico. By combining a systematic
deletion approach, coupled with analysis of the resultant mutants in refined models of
infection, we have begun to investigate the roles of unknown function genes of C. albicans
during interaction with the host.




                                                167
P113B
Pigment synthesis by Candida glabrata
Sascha Brunke and Bernhard Hube
Microbial Pathogenicity Mechanisms, Hans Knoell Institut, Beutenbergstr. 11a, Jena 07745,
Germany, Phone: +49 (0)3641 65 6885, FAX: +49 (0)3641 65 6882, e-
mail: sascha.brunke@hki-jena.de

Pigment synthesis is an important factor in the pathogenicity and virulence of many fungi. In
most cases, brown to black melanin is formed - for example in Aspergillus species,
Histoplasma capsulatum or Cryptococcus neoformans. In these organisms, melanin contributes
to the protection against UV light, chemical and mechanical stresses, oxidative killing by the
immune system and antifungal drugs. Recently, a new type of indole-derived pigment was
described for Malassezia furfur, a human skin pathogen. It could be shown that the same type
of pigment is also produced by Candida glabrata, a fungus with increasing importance in life-
threatening systemic candidiasis. Based on this finding, we started to investigate the possible
role of the pigment in pathogenicity and the genetic basis of its synthesis. By screening a
transposon insertion library of C. glabrata, kindly provided to us by Brendan Cormack, we
found several mutants of interest. Among these, some produce significantly less or no pigment
under pigment-inducing conditions, while others show an increased pigmentation. Sequencing
revealed that insertions took place independently at the same genetic loci in different mutants.
We concluded that these genes may be involved in the biosynthesis of the pigment. Using
these mutants, we started to investigate the role of the pigmentation in the life cycle of C.
glabrata. Preliminary experiments point towards a role in protection against oxidative agents,
which may contribute to survival in the host. Also, comparable to melanins, some protection
against UV irradation could be shown. Currently, these investigations are being extended to
include transcriptional profiling and examinations into the role of the pigment during
interactions with the host.




                                               168
P114C
Alternative carbon metabolism is required for virulence in Candida albicans and is
regulated by a unique mechanism.
Michael Lorenz, Melissa Ramirez, Aaron Carman and Huaijin Zhou
Microbiology and Molecular Genetics, The University of Texas Health Science Center, 6431
Fannin, Houston TX 77030, United States, Phone: (713) 500-7422, FAX: (713) 500-5499, e-
mail: Michael.Lorenz@uth.tmc.edu, Web: http://www.lorenzlab.org

The acquisition of nutrients is a primary concern of all microorganisms. In pathogens,
however, there have been relatively few studies examining the requirement for and regulation
of specific nutrient utilization pathways, yet it is increasingly clear that pathogens encounter
nutrient stresses within the host. For Candida albicans, the most important fungal pathogen of
humans, macrophage phagocytosis induces a transcriptional response strikingly similar to
carbon starvation, including a dramatic upregulation of numerous genes involved in the
production, transport, and use of acetate or acetyl-CoA, key intermediates in carbon
metabolism, such as acetyl-CoA synthases, hydrolases, acetyl-carnitine transporters, and the
glyoxylate cycle. We and others have demonstrated that several key carbon metabolic
pathways, notably ß-oxidation of fatty acids, the glyoxylate cycle, and gluconeogenesis are
both induced in vivo and required for full virulence in mouse models, as are some aspects of
acetate homeostasis. Surprisingly, in vitro phenotypes of mutations in ß-oxidation and the
glyoxylate cycle (via fox2 and icl1, respectively) differ from a priori expectations based on
studies in multiple other species. Specifically, the fox2 mutant strain is unable to use acetate,
lactate, citrate or ethanol as carbon sources; none of these compounds require the beta-
oxidation enzymatic machinery for assimilation. This indicates that the regulation of carbon
metabolism is significantly different in C. albicans compared to S. cerevisiae and other model
organisms. Consistent with this, we find that some well-characterized yeast carbon-source
transcriptional regulators are divergent or entirely missing in C. albicans, including ADR1,
OAF1, and PIP2. Instead, we have identified CTF1, a putative zinc-finger transcription factor
whose closest homologs are in filamentous fungi (farA and farB from Aspergillus). Mutants
lacking CTF1 are unable to grow on fatty acids, similar to the phenotype of the A. nidulans
farA farB double mutant. Thus it appears that C. albicans has adopted a hybrid regulatory
network, with some components resembling other yeasts and others more similar to
filamentous ascomycetes.




                                                169
P115A
cDNA representational difference analysis used in the identification of genes expressed by
Trichophyton rubrum during keratin contact
Lilian Cristiane Baeza1, Alexandre Melo Bailão2, Clayton Luiz Borges2, Maristela Pereira2,
Célia Maria Almeida Soares2 and Maria José Soares Mendes Giannini1
1
  Departamento de Análises Clínicas, UNESP - Universidade Estadual Paulista, Rua
Expedicionários do Brasil, 1621, Araraquara SP 14801-902, Brazil, Phone: +55 (16) 3301-
6556, FAX: +55 (16) 3301-6547, e-mail: baezalc@fcfar.unesp.br 2 Laboratório de Biologia
Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás, Goiânia, GO,
Brazil, CEP 74001-970

Dermatophytes are adapted to infect keratinized tissues by their ability to utilize keratin as a
nutrient source. Trichophyton rubrum is an anthropophilic fungus, causing up to 90% of
chronic cases of dermatophytosis. The understanding of the complex interactions between the
fungus and their host should include the identification of genes expressed during infection. To
identify genes involved in the infection process, the representational difference analysis (RDA)
was performed using two cDNA populations of T. rubrum, one obtained from fungus cultured
in the presence of keratin and other generated during fungal growth in minimal medium. Our
analysis identified transcripts differentially expressed. Genes related to signal transduction,
membrane protein, oxidative stress response, and some putative virulence factors were up-
regulated during the contact of the fungus with keratin. Interestingly, the expression patterns of
these genes was also verified by real-time PCR, in conidia of T. rubrum infecting in vitro
primarily cultured human keratinocytes, revealing its potential role in the infective process. A
better understanding of this interaction will contribute significantly to our knowledge the
infectious process of dermatophytes.

Keywords: Trichophyton rubrum, Representational difference analysis, Infection.




                                                170
P116B
In vitro and in vivo interactions of Candida albicans 14-3-3 mutants with host cells.
Michelle Kelly, Glen Palmer and Joy Sturtevant
Microbiology and Immunology, LSUHSC School of Medicine, 1901 Perdido St, New Orleans
LA 70117, USA, Phone: +1 504-568-6116, FAX: +1 504-568-2918, e-mail: jsturt@lsuhsc.edu

The opportunistic fungal pathogen Candida albicans can shift from a benign, commensal
organism to an invading pathogen, resulting in multiple clinical presentations from cutaneous
to life- threatening systemic candidiasis. This switch necessitates co-regulation of numerous
signaling networks, enabling Candida to evade host immune defenses, utilize new nutrient
sources, and set up successful infection. 14-3-3 proteins regulate many metabolic pathways.
The Candida 14-3-3 protein (Bmh1p) is required for multiple cellular functions including
vegetative growth, optimal filamentation, chlamydospore formation, response to the antibiotic
rapamycin, and carbon metabolism. Previously, a panel of isogenic strains was constructed via
site directed and random mutagenesis of the 14-3-3 gene (BMH1). Each of the mutant strains
has distinct phenotypes and thus each mutant is affected in a different subset of cellular
functions. We hypothesized that the bmh1 mutants would interact differently with host
cells. The ligand binding K51E 14-3-3 mutant was defective in killing macrophages
demonstrating that Bmh1p influences the phagocytic interaction. In addition to retarded
filamentous growth, our results indicated this mutant was affected in cell surface properties.
These phenotypes did not affect phagocytosis by the macrophage but did induce a differential
host response. While not susceptible to the murine macrophage cell lines used, the differential
response induced by the bmh1 mutant and increased phagocyte survival could influence the
outcome of infection in a whole animal model. Indeed in the disseminated murine candidiasis
model, the K51E mutant strain was avirulent (100% survival after 30 days). The bmh1 mutant
strains K51R and M125R were also attenuated in virulence but did not demonstrate altered
interactions with murine macrophage lines. These results imply that different cellular functions
associated with virulence are affected in our bmh1 mutants, highlighting the multiple roles of
14-3-3 in pathogenesis.




                                               171
P117C
Capsular enlargement reduces the susceptibility of Cryptococcus neoformans to free
radicals and antimicrobial peptides
Oscar Zaragoza1, Manuel Cuenca-Estrella1, Juan Luis Rodríguez Tudela1 and
Arturo Casadevall2
1
   Centro Nacional de Microbiología, Servicio de Micología, Instituto de Salud Carlos III,
Carretera Majadahona-Pozuelo, Km 2, Majadahonda, Madrid 28220, Spain, Phone: +34 91 822
3661, FAX: + 34 91 509 7034, e-mail: ozaragoza@isciii.es,
Web: http://www.isciii.es/htdocs/centros/microbiologia/servicios/micro_servicio_micologia.jsp
 2
   Albert Einstein College of Medicine, Bronx, New York

Cryptococcus neoformans is a unique human pathogen because it is the only encapsulated
fungus. The capsule is considered the main virulence factor (1) and contributes to virulence by
interfering with the immune response (see review in 2). A key aspect of the capsule is that its
size changes depending on the environment. The capsule is small in regular laboratory media.
However, several factors that induce a significant capsule enlargement have been described
(see review in 3). In addition, it is believed that capsule growth plays a role during
pathogenesis, since this morphological change is a response of the pathogen upon infection in
mice (4). The importance of capsule growth for cryptococcal pathogenesis is supported by the
fact that mutants that cannot enlarge the capsule show decrease virulence (5). Capsular growth
and rearrangements differ in various organs, suggesting a role in virulence (6). Although
capsule growth is a very characteristic feature in C. neoformans, its role during the interaction
with the host is poorly characterized. We hypothesized that capsule growth is an important
mechanism involved in the defence of the fungus against the multiple stress factors produced
by phagocytic cells. To test our hypothesis, we are measuring the viability of C. neoformans
cells with different capsule sizes exposed to different stress killing conditions. Our preliminary
results demonstrate that cells with enlarged capsule are more resistant to killing produced by
antimicrobial peptides (defensins) than cells with small capsule in a dose dependent manner.
We have also tested resistance to reactive oxygen species (ROS). While 0.5 mM H2O2
concentration killed more than 90% of cells with small capsule, cells with enlarged capsule
showed an increased viability (around 60% of killing). We are currently confirming our
hypothesis with other type of stresses, including primary phagocytic cells, and also
investigating the mechanism by which capsule growth produces resistance to killing by these
elements. The stress factors tested play a key role in pathogen killing by phagocytic cells.
Since C. neoformans is a facultative intracellular pathogen in macrophages, we believe that our
results suggests an explanation for the C. neoformans intracellular survival.

References
1. Inf. Immun. 2006. 74, 1500-04
2. Curr Mol Med. 2005. 5, 413-20
3. Biol Proced Online. 2006. 6, 10-15
4. Microbiol.2001. 147, 2355-65
5. J Clin Inv. 1985. 76, 508-516
6. Inf. Immun. 2004. 72, 3359-65




                                                172
P118A
AUF gene-cluster potentially involved in host-pathogen interaction
Martin Zavrel, Rosa Hernandez, Kai Sohn, Nicole Hauser and Steffen Rupp
MBT, Fraunhofer IGB, Nobelstrasse 12, Stuttgart 70569, GERMANY, Phone: +49 (0)711 970
4048, FAX: +49 (0)711 970 4200, e-mail: zav@igb.fraunhofer.de

Candida albicans is a commensal organism living on skin and mucosal surfaces of humans and
is also an important opportunistic pathogen. This yeast is able to adapt to various surfaces and
different host niches. One of the first steps in host-pathogen interaction is adhesion to the tissue
surface, an essential prerequisite for infection of the mammalian host. We have investigated the
response of C. albicans to different tissue using transcriptional profiling and could identify
genes differentially expressed on different surfaces or tissue. One of the ORFs upregulated
during the adhesion process is AUF8 (Adhesion Upregulated Factor), a gene without homolog
in Saccharomyces cerevisiae. The highest expression level appears after 120 minutes of the
adhesion process, with the strongest induction on the colorectal carcinoma Caco-2 cell-line
compared to the epidermoid vulvo vaginal cell-line A-431 or a polystyrene surface. The C.
albicans cells grown in suspension do not express AUF8, indicating that AUF8 may play a role
in host-pathogen interaction. In C. albicans genome, there are another six ORFs with sequence
homology to AUF8 of whom five (AUF2 - 6) are with AUF8 in a 10 kb gene-cluster and one
gene (AUF1) in different place in genome. All the predicted proteins are composed of about
200 - 250 amino acids and all seem to carry four transmembrane domains. There were four
homologous ORFs found in Candida dubliniensis, all of them in one gene-cluster. Using a
mutational approach to study these genes in C. albicans we attempt to uncover their role in
host-pathogen interaction.




                                                 173
P119B
Response of Candida albicans during the invasion of human epithelia
Rosa Hernandez, Michael Guenther, Kai Sohn, Nicole Hauser, Gaby Zelt and Steffen Rupp
MBT, Fraunhofer IGB, Nobelstrasse, 12, Stuttgart BW 70569, Germany,
Phone: +49(0)7119704048, FAX: +49(0)7119704200, e-mail: rhe@igb.fraunhofer.de,
Web: http://www.igb.fraunhofer.de/

The incidence of fungal infections has considerably increased worldwide due to opportunistic
pathogens such as Candida spp . The expression of specific factors that allow this fungus to
adhere to, and penetrate into many different host tissues, represents one of the most important
virulence factors in this human pathogen which cause a diverse range of infections. A better
understanding of the factors which influence the tissue invasion during the colonization of the
host will be a first step in preventing infections. Our host-pathogen interaction studies have
been performed on two different reconstituted tissue systems mimicking the interaction of C.
albicans with a enterocytic tissue model (colorectal adenocarcinomal cell line Caco-2) and
vaginal tissue model (epidermoid carcinomal cell line A-431)(1). To analyse the differential
gene expression in C. albicans during the processes of invasion in these host cells we have
used transcriptomic and proteomic approaches by isolating RNA and proteins from the
pathogen attach to and within host cells. To identify fungal genes that are differentially
regulated in C. albicans during the invasion of different epithelia we have performed
transcriptional profiling using self-developed C. albicans DNA-microarrays. In addition to a
common transcriptional response during the invasion of different epithelial cells, C. albicans
also has established individual adaptions when grown on specific tissues (Caco-2 cell line or
A431 cell line). We have also used combined proteomic strategies (2D-PAGE and mass
spectrometry) to detect and identify the differentially expressed proteins, as complementary to
the studies of the DNA-microarray studies. The study of the differential C. albicans protein
expression in these conditions has been performed by 2D-PAGE followed by SYPRO Ruby
staining. As a summary of preliminary results we can say that the initial transcriptomic and
proteomic studies of C. albicans during the invasion of different human tissue models resulted
in modifications of gene expression profile and proteomic patterns. The combination of these
two high throughput studies will certainly accelerate our knowledge of tissue-specific gene
expression in C. albicans and will therefore help us to understand why it is such a successful
fungal commensal and pathogen.

1. Sohn K. et al. 2006. FEMS Yeast Res. Nov;6(7):1085




                                               174
P120C
Comparative analysis of global gene expression in Candida dubliniensis and Candida
albicans in the reconstituted epithelial model of human oral candidosis
Martin J Spiering1, Karsten Hokamp2, Gary P Moran1, David C Coleman1 and Derek
J Sullivan1
1
  Microbiology Research Unit, Dublin Dental School & Hospital, Trinity College Dublin,
Lincoln Place, Dublin 2, Ireland, Phone: +353 (0)1 612 7260, FAX: +353 (0)1 6127295, e-
mail: spierinm@tcd.ie 2 Smurfit Institute of Genetics, Trinity College Dublin, Ireland

Candida dubliniensis is mainly associated with oral candidosis in HIV patients, but is far less
prevalent in other patient groups than its close relative, Candida albicans. Consistent with the
in vivo findings, C. dubliniensis shows lower virulence in the mouse systemic model, lower
rates of filamentation, and causes less damage to reconstituted human oral epithelium (RHE)
than C. albicans. However, the reasons for these differences are still not understood. In this
study, we compared growth, morphology, and global gene expression in C. dubliniensis (strain
CD36) and C. albicans (SC5314) in the RHE model of oral candidosis within 12 h post-
infection (p.i.). Candida albicans cells showed filamentation (formation of
hyphae/pseudohyphae) within 30 min p.i. and increased adherence and damage to the RHE
(assessed as the release of human lactate dehydrogenase (LDH) into cell culture medium)
within 4-8 h p.i. In contrast, at all time points, C. dubliniensis cells grew principally in the
yeast phase and exhibited little adherence and no significant damage to the RHE even at 12 h
p.i. Total RNA from Candida-RHE co-cultures was harvested at 30 min, 90 min, 6 h, and 12 h
p.i., along with controls, i.e., inoculum cultures and Candida cells incubated on polycarbonate
filters used as support matrix for the RHE. The C. albicans and C. dubliniensis RNAs were
used in co-hybridizations to Eurogentec C. albicans gene arrays, and expression data analysed
in ArrayPipe (at http://www.pathogenomics.ca/arraypipe/ ) to identify genes with statistically
significant (P<0.05, t-tests) differences in expression between the two species and among
treatments. Consistent with its filamentous phenotype on the RHE, C. albicans showed up-
regulation of hyphally regulated genes (e.g, ECE1, HYR1), and of cell-surface-associated genes
(e.g., PRA1) previously identified to be expressed in co-culture with human cells. Genes up-
regulated in C. dubliniensis included several ribosomal and metabolite-transport genes (e.g.,
HGT12), suggesting a less specialized response of C. dubliniensis to the RHE environment.
Detailed data analyses are underway to identify temporal patterns of gene expression in the two
species, to identify Candida genes involved in environmental sensing and signalling during
RHE colonization and subsequent infection.




                                               175
P121A
Virulence of Candida albicans recurrent isolates: a comparative analysis
Marlene Santos1, Paula Sampaio1, Alexandra Correia1, Gil Castro2, Jorge Pedrosa2 and
Célia Pais1
1
  Department of Biology, University of Minho, Campus de Gualtar, Braga 4710-057, Portugal,
Phone: 00351253604310, FAX: 00351253678980, e-mail: mmarlenes@gmail.com 2 Life and
Health Sciences Research Institute (ICVS), University of Minho, Campus de Gualtar, Braga,
4710-057 ,Portugal

Candida albicans infections are a major threat among immunocompromised patients and,
despite of the therapeutic approaches, reinfection episodes are relatively common. General
consensus exists that recurrences are due to the persistence of the original infecting strain and
cases of microevolution are well documented, reflecting strain adaptation. The objective of the
present work was to compare two C. albicans strains (A and B) isolated from a patient that
presented reinfection episodes, regarding their main phenotypic characteristics and their
virulence by using in vivo and in vitro approaches. These two isolates had the same multilocus
genotype, but the second one presented a loss of heterozygocity at one of the seven
microsatellite loci examined. The comparative analysis of colony morphology showed different
filament aggregation with a prevalence of the unicellular form in the original isolate (A).
Intracellular killing was accessed using the macrophage cell line J774 and data showed that
isolate A was able to form the germinative tube earlier that B, which is crutial to escape
intracellular killing by macrophages. To evaluate the degree of virulence of these two strains,
BALBc mice were infected systemically. Results showed that mice infected with isolate B
lived longer than those infected with strain A. Consistent with this observation was the fact that
mice infected with isolate A showed an increased fungal kidney burden. Histological analysis
showed a higher invasion of C. albicans in filamentous and non filamentous state in the kidney
of these mice, as well as an increase in inflammatory cells. This higher inflammatory infiltrate
is in agreement with the cytokine analysis, where there was a prevalence of IFN-gamma and
TNF-alfa in mice infected with isolate A. The elevated levels of inflammatory cytokines
together with the massive kidney colonization might be responsible for the death. Herein we
show that selective pressure inducing C. albicans microevolution may result in reduced
virulence, favouring the persistence of the strain in the host. This research was supported by
the grant POCTI/SAU-IM/58014/2004 from FCT (Fundação para a Ciência e Tecnologia.)




                                                176
P122B
Characterization of Pga29p, an abundant cell wall protein of the human pathogen
Candida albicans that mediates virulence in a RHE model of oral candidosis.
Albert de Boer1, Piet de Groot2, Martin Schaller3, Günther Weindl3, Frans Klis2, Uwe Gross1
and Michael Weig1
1
  Medical Microbiology, University of Göttingen, Kreuzbergring 57, Göttingen 37075,
Germany, Phone: +49 (0) 551 39 5848, FAX: +49 (0) 551 39 5861, e-mail: aboer@gwdg.de 2
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166,
1018 WV Amsterdam, The Netherlands, Phone: +31 (0) 20 525 7834, FAX: +31 (0) 20 525
7056, e-mail: P.W.J.deGroot@uva.nl 3 Dermatology, University of Tübingen,
Liebermeisterstrasse 25, 72076 Tübingen, Phone +49 (0) 7071 2984555, FAX: +49 (0) 7071
295113, e-mail: Martin.Schaller@med.uni-tuebingen.de

Covalently linked cell wall proteins (CWPs) of the dimorphic human pathogenic fungus
Candida albicans play an important role in host-pathogen interactions that may lead to fungal
infections. One of the most abundant CWPs in C. albicans yeast cell wall is
Pga29p/Rhd3p/orf19.5305, a small GPI-modified glycoprotein of about 30 kDa. During hyphal
growth it is downregulated (Nantel et al., 2002), while increased expression of the gene is
observed in protoplasts regenerating their cell wall (Castillo et al., 2006). Apart from closely
related pathogenic Candida species, no homologues of Pga29p are found in other fungi. This
urged us to further characterize the function of this abundant CWP. Immuno-blot analysis
showed that this protein is abundant in cell walls that were purified from yeast grown at 30°C,
whereas it is undetectable at 37°C and 42°C. Cell wall fractions from cells that were exposed
to the cell wall perturbant Calcofluor White (CFW) during growth showed a strong increase of
Pga29p incorporation, indicating that C. albicans cells may stabilize their cell wall by
upregulating Pga29p levels. In order to explore the function of Pga29p more deeply, we
generated pga29/pga29 mutants. Deletion of PGA29 resulted in an increased glucose/mannose
ratio in the cell wall. However, no difference in sensitivity was observed between mutant,
reconstituted and wild type strains under several cell wall stress conditions (CFW, osmolarity,
SDS and enzymatic glucan digestion), suggesting that Pga29p has a function other than a
major structural entity in the cell wall of C. albicans. In an in vitro model of oral candidosis
based on reconstituted human epithelium (RHE), pga29/pga29 mutants showed a clear
reduction in virulence compared with reconstituted and wild type strains. Additionally, we
studied the immune response of the epithelial cells against our strains by quantifying mRNA
levels for cytokines. Epithelial cells that were infected with the mutant strain showed
significantly reduced expression levels of GM-CSF, TNF-alpha and IL-8. This suggests that
Pga29p has an important role in mediating oral candidosis. To elucidate whether Pga29p is
directly or indirectly involved in the infection process, we are currently infecting RHE with a
mutant that is overexpressing PGA29. Additionally, we investigate the immune response of
RHE incubated with purified cell walls and cell wall protein extracts of the mutant,
reconstituted and wild type strains.




                                               177
P123C
Genetic diversity of Pneumocystis jirovecii and interaction with the human host
Jessica Beser, Silvia Botero, Marianne Lebbad, Per Hagblom and Victor Fernandez
Parasitology, Mycology and Environmental Microbiology, Swedish Institute for Infectious
Disease Control, Nobels väg 18, Solna 17182, SWEDEN, Phone: +46 (0)8 4572544, FAX: +46
(0)8 318450, e-mail: jessica.beser@smi.ki.se, Web: www.smittskyddsinstitutet.se

Pneumocystis jirovecii is an atypical fungus with a worldwide distribution that causes disease
in immunocompromised persons. The fungus proliferates in the lungs where it binds
specifically to type-1 epithelial alveolar cells provoking severe pneumonia, denoted
Pneumocystis pneumonia (PCP). For a long time it was thought that disease was caused by
reactivation of latent Pneumocystis infection, now there is evidence that person-to-person
transmission and reinfection commonly occurs. The fungus can be detected in healthy
individuals indicating that humans are a possible reservoir as well as a source for transmission.
To study the epidemiology of PCP it is necessary to identify different strains of P. jirovecii. In
the course of measuring genetic diversity in the local P. jirovecii population by analysis of the
ITS locus, we optimized the most commonly used method for epidemiological typing of this
fungus (Beser et al., (2007), J. Clin. Microbiol., 45). This improved tool has enabled a more
correct assessment of the previously overestimated genetic diversity of P. jirovecii populations.
At the individual level it has now become possible to address with precision questions
concerning burden of infection, transmission and reinfection. One molecular player of P.
jirovecii with a probable key function in the colonization of the alveoli and immune evasion is
the major surface glycoprotein (MSG) encoded by the msg genes. The MSG is the most
abundant protein expressed on the surface of P.jirovecii and appears to act as an attachment
ligand to the alveolar pneumocytes. The MSGs are encoded by a multicopy gene family with
up to 100 isoforms spread on all the chromosomes in the genome. Transcription is probably
limited to a single MSG gene translocated to a unique expression site. This mechanism
probably facilitates antigenic variation, which is a mean for the microbe to evade the host’s
immune system. With the aim of understanding how the fungus controls the exposure of this
large antigenic and ligand repertoire, we are currently studying diversity at the locus associated
with msg transcription. Additionally, these studies which are carried out in respiratory
specimens well characterized with respect to the degree of heterogeneity at the ITS locus, shed
valuable information on the population structure of P. jirovecii and dynamics of infection.




                                                178
P124A
Changes in adherence but not virulence of fluconazole-resistant Candida albicans
Bettina Schulz, Kai Weber, Michael Fleischhacker and Markus Ruhnke
Molekulare Infektionsdiagnostik, Charité Universitätsmedizin Berlin, Charitéplatz 1, Berlin
10117, Germany, Phone: +49 (0) 30 450 513376, FAX: +49 (0) 30 450 513976, e-
mail: b.schulz@charite.de, Web: www.charite.de

The development of fluconazole (FLU) resistant C. albicans strains during the treatment of
oral and oesophageal candidiasis in patients with AIDS has been frequently described. To date,
it is not known whether the development of FLU resistance affects the virulence of these
strains. The objective of this study was to examine various genes which were contributed to be
important for virulence of C. albicans and whether these findings differ between FLU sensitive
and FLU resistant strains. Five pairs of C. albicans isolates (N=10) from five different HIV-
positive patients with FLU-refractory oral candidiasis were selected for this study. Pairs of C.
albicans isolates were taken from the first episode of oral candidiasis (FLU sensitive) as well
when patients developed refractory candidiasis and the isolates were resistant to FLU. The
obtained strains were subcultured in RPMI 1640 supplemented with fetal calf serum to induce
hyphal growth. For comparison the isolates were also cultured in YPD-medium keeping the
cells in the blastospore phenotype. RNA isolation was performed of both growth forms and
followed by cDNA synthesis and gene expression analysis using Real-Time PCR. As virulence
genes were examined different members of the secreted aspartyl protease (SAP) family (SAP2,
SAP5, SAP8, SAP9), CSH1 coding for a protein which influences the cell surface
hydrophobocity and TUP1, a gene negatively regulating filamentous growth. Additionally, the
genes of the C. albicans efflux pumps CDR1 and CaMDR1 were included. Gene expression
was increased for the FLU-refractory isolates either for CDR1 or CaMDR1 confirming their
resistant state. The gene expression analyses of SAP genes revealed no consistent pattern
between susceptible and resistant strains and no clear differences between blastospores and
hyphae. The expression profile of TUP1 showed only the growth form dependent regulation,
being upregulated in blastospores and downregulated in hyphae and no differences between
FLU sensitive and FLU resistant strains. In contrast, CSH1 expression was upregulated in
resistant isolates growing in the blastospore phenotype indicating a higher adhesion capacity of
blastospores in comparison to hyphae. These findings suggest that resistance has not to be
necessarily the reason for higher virulence but differences in adherence properties could
promote an infection of C. albicans.




                                               179
P125B
How to get Candida albicans plasma membranes for differential proteomic analysis.
Virginia Cabezón, César Nombela and Concha Gil
Microbiology II, Faculty of Pharmacy. UCM, Plaza Ramón y Cajal s/n, Madrid 28040, Spain,
Phone: +34913941755,           FAX: +34913941745,        e-mail: vcabezon@farm.ucm.es,
Web: www.ucm.es

Candida albicans is a dimorphic opportunistic human pathogen. It causes a wide variety of
infections ranging from mucocutaneous infection to deep systemic syndromes. We have
carried out a proteomic approach of the C. albicans-macrophage interaction using total extracts
(1) where we provide evidence of a rapid protein response of the fungus to adapt to the new
environment inside the phagosome by changing the expression of proteins belonging to
different important pathways related to Candida’s virulence. Our next aim is to study the
differential expression of the plasma membrane proteins because they play an important role in
signal trasduction, cell adhesion, ion transport, in the regulation of dimorphism and virulence
and they are important pharmacological targets. Because of their hydrophobic and basic nature
and frecuently large size, their isolation is not easy (2). After trying different strategies to get a
plasma membrane enriched fraction, we are setting up a protocol based on protoplasts
generation and lysis, use of a sucrose gradient and sodium carbonate treatments in order to
convert closed vesicles in open membranes sheets (3) and avoid contamination with
cytoplasmatic proteins. Membrane proteins were analysed using two approaches. In the first
one proteins were separated by SDS-PAGE and the bands were cut, digested and analysed
using a MALDI-TOF-TOF. In the second approach solubilized proteins were digested and
analysed by LC-MS-MS and a total of 32 proteins previously identified as plasma membrane
proteins.

(1) E. Fernández-Arenas, V. Cabezón, C. Bermejo, J. Arroyo, C. Nombela, R. Díez-Orejas,
C.Gil. Integrated proteomic and genomic strategies bring new insight into Candida albicans
response upon macrophage interaction. Molecular and Cellular Proteomics (Dic 2006).
(2) M. W. Qoronfleh, B. Benton, R. Ignacio and B. Kaboord. Selective enrichment of
membrane proteins by partition phase separation for proteomic studies. Journal of Biomedicine
and Biotechnology 2003:4 (2003) 249-255.
(3) Yukio Fujiki, Ann L. Hubbard, Stanley Fowler, and Paul B. Lazarow. Isolation of
intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic
reticulum. The journal of cell biology. Vol. 93 April 1982 97-102.




                                                  180
P126C
Sexual development in Cryptococcus neoformans is modulated by the steady-state
concentration of endogenous reactive oxygen species
Steven Giles and Christina Hull
Biomolecular Chemistry, University of Wisconsin-Madison, 687 Medical Sciences Center,
1300 University Ave., Madison WI 53562, USA, Phone: (608) 265.5689, FAX: (608)
262.5253, e-mail: ssgiles@wisc.edu, Web: http://www.bmolchem.wisc.edu/faculty/hull.html

Cryptococcus neoformans (Cn) utilizes antioxidant defense mechanisms for protection against
oxidative damage during stressful processes such as adaptation to aerobic environments,
dependence on oxidative phosphorylation for energy production, and protection against
reactive oxygen species (ROS) encountered in the environment. In previous studies we have
demonstrated that Cn has a robust antioxidant defense system, which includes four catalases.
Unilateral crosses with cat4 mutants resulted in normal sexual development. However, bilateral
crosses resulted in impaired sexual development. We hypothesize that Cat4 provides
antioxidant defense during sexual development. To assess the role of catalases in sexual
development unilateral and bilateral crosses were performed with various Cn catalase mutant
strains. Crosses were performed in the presence of exogenous antioxidants to determine if the
steady-state concentration of endogenous ROS affected sexual development. We confirmed
that only bilateral crosses with cat4 mutants resulted in impaired sexual development.
Although dikaryotic filaments with chains of basidiospores were observed, the majority of
filaments were imbedded in the agar, as opposed to projecting from the agar and forming the
aerial filaments with spore chains observed during wild-type crosses. Given that cells are in a
state of nutrient deprivation during sexual development, we would predict that this process
would result in an increase in the steady-state concentration of endogenous ROS. If the role of
Cat4 in sexual development is to regulate the steady-state concentration of endogenous ROS,
then we would also predict that the cat4 mutant phenotype would be complemented by addition
of exogenous antioxidants. However, the opposite effect was observed. We found that the
addition of exogenous antioxidants (ascorbic acid) completely abrogated sexual development
of Sero A strains. In contrast, ascorbic acid had no affect on the sexual development of Sero D
strains. Microarray analysis is currently underway to identify genes that are differentially
regulated in Sero A strains during sexual development in response to changes in the steady-
state concentration of endogenous ROS. These results suggest that sexual development in Sero
A, but not Sero D strains of Cn is regulated in response to endogenous ROS levels. In addition,
these results suggest that these closely related serotypes have evolved divergent pathways to
initiate sexual development.




                                               181
P127A
Co-ordinate regulation of A. fumigatus-specific gene clusters involved in secondary
metabolite biosynthesis, macromolecule catabolism and transmembrane transport during
initiation of infection
Andrew McDonagh1, Natalie Fedorova2, Yan Yu2, Stanley Kim2, Darius Armstrong-James1,
Ken Haynes1, William Niermann2 and Elaine Bignell1
1
  Department of Molecular Microbiology and Infection, Imperial College London, Armstrong
Road, London SW72AZ, United Kingdom, Phone: 0044 207 594 2074, FAX: 0044 207 594
3076, e-mail: e.bignell@imperial.ac.uk 2 The Institute for Genomic Research, Rockville, MD

Aspergillus fumigatus is the Earths most harmful mould, causing both fatal invasive infection
and severe allergic disease. The molecular basis of A. fumigatus pathogenicity remains a
puzzling conundrum because the metabolic versatility required for its predominantly
saprophytic lifestyle necessarily bestows the fortuitous support of growth on diverse substrates,
including lung tissue, the effect of which is not easily separable from so-called true virulence
traits. With the notable exceptions of siderophore biosynthesis and global regulation of
Aspergillus secondary metabolite biosynthesis the candidate gene approach to identification of
virulence determinants has met with marginal success, a favourable explanation for which is
the requirement for precise orchestration of multiple functions in order to cause disease. To
understand the A. fumigatus response to the host environment at an early stage of infection we
compared the transcriptome of developmentally matched A. fumigatus isolates following
culture in vitro or rescue from the neutropenic murine lung, with the broad aim of assessing
whether transcriptional differences are: a) largely metabolic and nutritional, supporting the
notion that the organism is an accidental pathogen, or b) specifically involved in pathogenicity,
toxicity and immune evasion. To achieve this we developed an mRNA amplification protocol
to overcome the practical difficulties associated with recovery of A. fumigatus RNA from the
bronchoalveolar lavage fluid (BALF) of infected mice. We assessed the maintenance of
relative transcript abundances following RNA amplification using an engineered lithium
chloride exposure experiment and applied linear modelling to identify transcripts whose
reported log ratios represent a biological effect rather than systematic bias. Multiple hypothesis
testing was used to identify spots where a significant amplification protocol effect on log ratio
occurred. This approach yielded highly reproducible insights to A. fumigatus transcriptional re-
programming during initiation of invasive disease (compared with that in vitro) revealing
secondary metabolism, macromolecule catabolism and cellular transport among the weapons
deployed to initiate mammalian infection.




                                                182
P128B
Identifying virulence factors in the fungal pathogen Histoplasma capsulatum.
Dervla Isaac and Anita Sil
Microbiology and Immunology, University of California - San Francisco (UCSF), 513
Parnassus Ave, Room S-472, San Francisco CA 94143, United States, Phone: 011-1-415-502-
4810, FAX: 011-1-415-476-8201, e-mail: Dervla.Isaac@ucsf.edu

Histoplasma capsulatum is thought to be the most common cause of fungal respiratory
infections. This intracellular pathogen infects both humans and mice, surviving within the
hostile environment of host macrophages. Macrophages are immune cells that ingest and digest
invading microbes. However, H. capsulatum replicates within macrophages, ultimately
resulting in lysis of the host cell. We have successfully designed a genetic screen to identify H.
capsulatum mutants that fail to lyse murine bone marrow derived macrophages. A screen of
5,000 H. capsulatum insertion mutants identified seven lysis defective mutants. These mutants
lyse macrophages with moderately to severely delayed kinetics compared to wildtype, but do
not exhibit any defects in intracellular growth. One ldf mutant contained an insertion in the
promoter of a MYB DNA binding protein homologous to S. cerevisiae TBF1, telomere binding
factor 1. Since MYB DNA binding proteins are known to regulate gene expression, we
hypothesize that the H. capsulatum TBF1 activates a gene (or genes) that is required to
colonize macrophages appropriately. Lysis of macrophages infected with the tbf1 mutant is
drastically compromised, as demonstrated by measuring macrophage monolayer integrity and
LDH release during the course of the infection. In addition to this severe lysis defect, tbf1
infected macrophages adopt an abnormally vacuolated morphology during infection. We are
currently using microarrays to compare the gene expression profiles of wild-type and tbf1
mutants in macrophages. Other genes identified in this screen include an HMG CoA lyase
(HCL1) and several predicted genes with no homologs in other organisms. Further
characterization of these mutants is ongoing to determine how they respond to and manipulate
host macrophages.




                                                183
P130A
Comparison of gene expression in Candida albicans and Candida dubliniensis in human
serum
Nicole Caplice, Derek Sullivan, David Coleman and Gary Moran
Microbiology Research Unit, Division of Oral Biosciences, Dublin Dental School and
Hospital, Lincoln place, Dublin D 2, Ireland, Phone: +353 (0)1 612 7350, FAX: +353 (0)1 612
7295, e-mail: nicole.caplice@dental.tcd.ie

Candida species are the fourth most common cause of hospital acquired bloodstream
infections, with an attributable mortality of up to 35%. C. albicans is responsible for 60% of
Candida bloodstream infection’s, whereas it’s close relative C. dubliniensis is accountable for
only 2%, the reason(s) for which are at present unclear. As the genome sequences of both
species are highly similar we hypothesise that inter-species variation in gene expression could
account for the virulence differences exhibited by both species. The aim of this study was to
compare hyphal formation, yeast cell viability and expression of hyphal specific genes in C.
albicans and C. dubliniensis in human serum. The percent of hyphal formation of both species
in 50% human serum at 37°C was estimated. After 2 hours >90% of the C. albicans cells
formed hyphae, while approximately half of the C. dubliniensis cells were still in yeast phase.
Staining with FUN1 dye and fluorescence microscopy revealed that incubation in serum for 3
hours did not affect cell viability. GFP reporter systems were constructed to investigate hyphal
specific gene expression in both Candida species at the level of the single cell. The integrating
vector contained a yEGFP reporter gene fused to either an (ACT, ECE1 or HWP1 promoter), an
ACT terminator and SAT1. Both Candida species were transformed by electroporation and
resistant transformants were selected. These strains were induced to produce hypha in 10% and
50% human serum and examined by fluorescence microscopy and flow cytometry at different
time intervals. Preliminary flow cytometry data generated from C. albicans ECE1
promoter/GFP fusion strains incubated in 10% and 50% serum for 2 hours revealed a 25 fold
and 13 fold increase in mean fluorescence intensities respectively while the C. dubliniensis
strain incubated under the same conditions did not display any significant difference. In
conclusion, C. dubliniensis is less able to form germ tubes in response to serum, which would
significantly affect its ability to disseminate in vivo. Patterns of gene expression in these two
species and the Nrg1 mutant will be examined further both at the level of the single cell using
promoter-GFP reporter fusions and globally using micro-arrays. The effects of pH, temperature
and different (serum, glucose and farnesol) concentrations on the hyphal specific gene
expression of these Candida species will also be investigated.




                                                184
P131B
Contribution of mannoproteins to Candida albicans cell wall surface expression of
virulence determinants, the ß-1,2 oligomannosides
Chantal Fradin, Céline Mille, Pierre André Trinel and Daniel Poulain
Inserm U799, Faculté de médecine, Place de Verdun, Lille 59045, FRANCE, Phone: + 33 (0) 3
20 62 34 21, FAX: + 33 (0)3 20 62 34 16, e-mail: cfradin@univ-lille2.fr

Surface mannoproteins have specific functions some of which being related to C. albicans
virulence like adhesion to host tissues and plastics, biofilm formation and immune system
triggering. Studies using mutants affected in their protein glycosylation, have shown that some
virulence attributes may be conferred to proteins through their N- and/or O-glycans. Emphasis
has been made on the role of fungal cell wall glycans either as polysaccharides or conjugated
to proteins or lipids in maintaining cell shape and integrity. However for the hosts, all these
fungal glycans represent non-self molecules. For C. albicans and namely in relation with the
commensal/pathogen transition several studies have focused on oligomannosides with an
unusual type of linkage. They consist in ß-1,2 linked oligomannosides (ß-Mans) that have been
shown to mediate the adhesion of C. albicans to host cells, induce cytokines production and
can generate protective antibodies. If the role of ß-Mans in the virulence of C. albicans has
been clearly established, little is known about their distribution and anchoring in the cell wall
and how they could be presented to the host. The objective of our study was to analyze if, in
addition to the phosphopeptidomannan and phospholipomannan for which the presence of ß-
Mans have been chemically established, other cell wall glycoconjugates could be ß-1,2
mannosylated and how ß-Mans are attached to these molecules. Using specific monoclonal
antibodies in Western Blot, we could demonstrate that ß-Mans are carried by mannoproteins
either bound covalently or not to C. albicans cell wall. After chemical and enzymatic
treatments, we could show that these oligomannosides are part of both N- and O-glycans. If
their presence in the N-glycans is a new information coherent with what has been chemically
for the phosphopeptidomannan, according to the literature, ß-1,2 mannosylation of C. albicans
O-glycans is more surprising. NMR analysis confirmed this observation and gave new insights
into the O-glycosylation processes. Using proteomic analysis, some of these mannoproteins
could be identified. Interestingly, some of them are i) expressed at the cell wall surface and/or
ii) known to contribute to C. albicans virulence. Presence of ß-Mans in the glycan moieties of
some C. albicans cell wall mannoproteins can confer either new or additional virulence
attributes to these proteins and be part of the certainly complex ß-1,2 mannosylation regulation
mechanisms.




                                                185
P132C
Identification of a new family of genes involved in biosynthesis of Candida albicans
virulence factors, the ß-1,2 oligomannosides
Céline Mille1, Pierre andré Trinel1, Chantal Fradin1, Guilhem Janbon2 and Daniel Poulain1
1
  Inserm U799, Faculté de médecine, place de verdun, Lille 59000, FRANCE, Phone: +33 (0) 3
20 62 34 15, FAX: + 33 (0)3 20 62 34 16, e-mail: celine.mille@univ-lille2.fr 2 Institut Pasteur,
Paris

Among the established factors contributing to C. albicans virulence are cell wall surface ß-1,2
oligomannosides. During C. albicans infection, ß-Mans play an important role in host/pathogen
interactions by acting as adhesins and by interfering with the host immune response. They have
been found to be associated with the cell wall PPM, PLM and more recently
mannoproteins. We have identified a family of genes designated CaBMTs involved in C.
albicansß-1,2 mannosylation. These genes have homologs in other Candida species, among
which C. glabrata and C. parapsilosis. Homologs are also present in Debaryomyces hansenii
and Pichia pastoris. Even if carrying ß-Man, this last specie is not pathogenic so that this
condition is not either necessary nor sufficient for being pathogenic. However, even if so far no
ß-Mans have been found in Aspergillus fumigatus, CaBMTs homologs are present in the
genome of this other opportunistic pathogenic fungus. The ability to construct ß-Mans is not
restricted to fungi as their presence has been chemically demonstrated in pathogenic bacteria,
namely some Salmonella serotypes as well as in protozoan parasites of the genus Leishmania.
The BMTs genes seem to be evolutionary distinct between these organisms and the fungi as no
CaBMTs homolog has been found in their genomes. Several reviews have demonstrated a
common origin and conserved domains of mannosyltransferases related to the specificity of
acceptor substrates. CaBmtps have strong homologies according to their functions which have
been characterized by combined immunochemical and structural studies on glycoconjugates of
the corresponding deletion strains. CaBMTs encode for ß-1,2 mannosyltransferases acting
specifically on either PPM, PLM or mannoproteins. Putative compensatory activities could
nevertheless be evidenced between CaBmtps involved in the same ß-1,2 mannosylation step
but on different acceptor molecules. Individual deletions of these genes have no impact on
global surface expression of ß-Mans and virulence. These results are coherent with the
diversity of glycoconjugates carrying ß-Mans at the cell wall surface and the substrate
specificity of the different CaBmtps. The relative role of ß-Mans in pathogenesis will only be
evidenced by using multiple deletion strains. In agreement with other studies, the work
presented here highlights a complex mechanism of ß-1,2 oligomannosides expression and
suggests the existence of a regulation process controlling the amount of ß-Mans at the cell wall
surface.




                                                186
P133A
A systematic approach reveals new virulence factors in Candida albicans.
Suzanne Noble, Lisa Kohn and Alexander Johnson
Microbiology and Immunology Department, UCSF, 600 16th St., Box 2200, San Francisco CA
94143-2200, United States, Phone: (001) 415-502-0859, FAX: (001) 415-502-4315, e-
mail: Suzanne.Noble@ucsf.edu

Candida albicans is the major cause of human fungal infections worldwide, yet our
understanding of its virulence mechanisms and options for treatment remain limited. To
approach the problem of C. albicans virulence using forward genetics, we have developed
methods to create homozygous knockout mutants in a high throughput manner (Noble and
Johnson, 2005). We have created approximately 800 signature-tagged homozygous deletion
mutants of C. albicans in which ORF sequences have been precisely replaced with selectable
auxotrophic markers that are themselves neutral for virulence. We are currently systematically
screening the mutant strains in vivo and in vitro for virulence-related properties including the
ability to successfully infect experimental mice, morphogenesis, and fluconazole resistance.
Results to date include the identification of previously undescribed predicted C. albicans cell
wall proteins as well as a number of presumptive regulatory proteins that are important for
infection of the host but dispensable for morphological transitions in vitro.

Noble and Johnson (2005). “Strains and strategies for large-scale gene deletion studies of the
diploid human fungal pathogen Candida albicans.” Eukaryotic Cell 4: 298-309.




                                               187
P134B
Hyphae of Candida spp drive sustained MAP kinase signaling through ERK
phosphorylation in primary macrophages and myeloid dendritic cells
Olivia Majer1, Ingrid Frohner1, Christelle Bourgeois1, Steffen Rupp2 and Karl Kuchler1
1
  Medical Biochemistry, Max F. Perutz laboratories, Dr. Bohrgasse 9/2, Vienna 1030, Austria,
Phone: +43 1 4277 61812, FAX: +43 1 4277 9618, e-mail: olivia.majer@meduniwien.ac.at 2
IGB Fraunhofer-Institute for Interfacial Engineering and Biotechnology, Nobelstraße 12, D-
70569 Stuttgart, Germany

The fungal pathogen Candida albicans (C.a) displays considerable morphogenetic plasticity.
The organism can grow in either yeast or hyphae form, or as physically intermediate forms
such as pseudohyphae. The ability of C.a to switch from the yeast to filamentous forms
through germ tube formation is considered a key feature of the transition of the organism from
commensalisms to virulence. This study aimed at investigating whether C.a yeast and hyphae
forms interfere differently with cells of the mouse innate immune system. Particular attention
was given to the ERK pathway that, in conjunction with other signaling pathways such as the
stress-activated protein kinase p38 pathway and NFkappaB signaling, play prominent roles in
the initial response and propagation of inflammation. To perform these in vitro host-pathogen
interaction studies, we used mouse bone marrow-derived primary macrophages (BMDMs) or
myeloid dendritic cells (mDCs). Mouse host cells were incubated with either mutants of C.a
trapped in the yeast form (efg1delta/cph1delta), those existing as constitutive hyphae
(tup1delta) or UV-treated Candida cells. We showed that Candida spp interaction triggered
rapid phosphorylation of ERK1/2 in mouse BMDMs and mDCs. Activation seemed to follow a
“2-phase induction”, depending on the morphological state of the fungus. The first phase
occurred within 30 minutes of C.a exposure and appeared to be independent of morphogenetic
state. The second phase, observed after 2 hours of interaction, was clearly morphology-
dependent, since only the hyphae form was able to sustain ERK1/2 signalling. By contrast,
interaction with the yeast form of C.a was unable to sustain long-lasting ERK phosphorylation.
In agreement with this data, in cells infected with Candida glabrata, a second major fungal
pathogen, which in contrast to C.a is only found in yeast form, ERK showed a phosphorylation
pattern similar to the yeast form of C.a. These results may reflect the qualitative and
quantitative differences in the pathogenicity of Candida glabrata and Candida albicans cells.




                                              188
P135C
The environmental dimensionality controls the interaction of phagocytes with the
pathogenic fungi Aspergillus fumigatus and Candida albicans.
Priyanka Narang1, Mike Hasenberg1, Judith Behnsen2, Axel Brakhage2 and Matthias Gunzer1
1
  Junior Research Group Immunodynamics, Helmholtz Zentrum für Infektionsforschung,
Inhoffenstrasse 7, Braunschweig 38124, GERMANY, Phone: +49 (0) 531 6181 3132,
FAX: +49 (0) 531 6181 3199, e-mail: pna04@helmholtz-hzi.de, Web: http://www.helmholtz-
hzi.de 2 Hans Knoll Institute, Jena, Germany

Aspergillus fumigatus and Candida albicans are two major fungal pathogens, which have
gained importance in recent years due to an increase in the number of immunocompromised
individuals affected by them. While A. fumigatus is an airborne pathogen, and is first
encountered by the resident Alveolar Macrophages (AMs) in the lung, and the recruited
polymorphonuclear cells (PMNs), C. albicans is an opportunistic pathogen associated with the
mucosal tissue, and is attacked mainly by the tissue resident PMNs. If immune control is lost,
both fungi grow into the surrounding tissue and cause life-threatening invasive diseases.
Although these pathogens have been widely studied, the cellular dynamics during the early
phase of infection are largely correlative. In addition, the lung provides two distinct
environments: a 3-D tissue environment surrounding the alveolar space, and a 2-D
environment within the alveoli, where the AMs are suspended in a very thin surfactant layer
close to the epithelial cells. It is not known whether and how this structural difference in the
environment affects the phagocytic ability of the cells. To investigate how phagocytes function
in these disparate environments of lung air sacs or mucosal tissues (providing a three-
dimensional [3-D] space), we mimicked 2-D (provided by media) and 3-D (collagen matrix)
environments and analyzed the process of ingestion called phagocytosis by PMNs and other
phagocytes by time lapse video microscopy. Phagocytosis was a dynamic cellular process
where distinct cells showed vastly different behavior. The environmental setup (2-D versus 3-
D) had a profound impact on the cell’s ability to phagocytose. Aspergillus conidia were much
better ingested in 2-D systems, while Candida yeasts were only ingested in 3-D systems, even
if the other pathogen was present. This was true for different 2-D and 3-D systems and for cells
of both mice and humans. Besides providing a comprehensive analysis of the cellular
movements underlying phagocytosis, the results also suggest an evolution of phagocytes to
optimally recognize fungal pathogens in the environment of natural infection.




                                               189
P136A
A strain of Aspergillus fumigatus expressing YFP and a model antigen for the study of
antifungal T cell responses
Mike Hasenberg1, Priyanka Narang1, Judith Behnsen2, Axel Brakhage2, Philipp Schmalhorst3,
Francoise Routier3, Sven Krappmann4 and Matthias Gunzer1
1
  Nachwuchsforschergruppe Immundynamik, Helmholtz Zentrum für Infektionsforschung,
Inhoffenstraße 7, Braunschweig 38124, Germany, Phone: +49 (0) 531 6181 3132, FAX: +49
(0) 531 6181 3199, e-mail: mike.hasenberg@gbf.de, Web: http://www.helmholtz-
hzi.de/de/forschergruppen/zell_und_immunbiologie/immundynamik/ 2 Leibniz-Institute for
Natural Product Research and Infection Biology (HKI); Beutenbergstrasse 11a; 07745 Jena,
Germany 3 Medizinische Hochschule Hannover, MHH ; Abteilung Zelluläre Chemie; Carl-
Neuberg-Strasse 1; 30625 Hannover, Germany 4 Georg-August-Universität Göttingen; Institut
für Mikrobiologie und Genetik; Abt. Molekulare Mikrobiologie und Genetik; Grisebachstr. 8;
37077 Göttingen, Germany

Aspergillus fumigatus is an opportunistic fungus that afflicts immunocompromised people
where it can lead to the often fatal Invasive Aspergillosis. While it is well known that the
innate immune system, particularly alveolar macrophages and neutrophil granulocytes,
constitute the major part of the immunological defence against this fungal pathogen, also cells
of the adaptive immune system have been implicated in response to Invasive Aspergillosis. To
clarify the roles of T cells in the time course of an infection we have generated an Aspergillus
strain expressing the fluorescent protein Citrin (EYFP) and Ovalbumin (Ova) as model antigen
for T cell activation. In vitro dendritic cells are able to take up and present this Aspergillus
strain to Ova specific T cells inducing their proliferation. This strain also shows intensive YFP
fluorescence which can be detected by 2-photon microscopy in infected tissues. Currently we
are using a murine infection model with the transgenic fungus to elucidate the time course of
the T cell response against Aspergillosis in vivo. To address this point we adoptively transfer
CFSE stained Ova specific T cells into recipient mice and infect these animals intranasally
with the transgenic or control Aspergillus strain. At different time points after infection we
analyze all major lymphatic tissues along the infection route for proliferating T-cells. In
ongoing work we evaluate the possibility of vaccinating mice against Aspergillus or provide
specific T cells. To get deeper insights into ongoing immune responses in infected tissues (i.e.
Lung) we are using 2-photon microscopy to visualize these locations. This approach should
highlight the cellular dynamics underlying the infection defence and how the message that an
in-fection is going on is transferred to immunological organization centers i.e. lymph nodes. In
summary we hope to get a better understanding of the cellular processes underlying the
generation of an Aspergillus specific immune response as well as the potency of this response
to protect from this lethal infection.




                                                190
P137B
Humoral immune response in invasive and allergic aspergillosis mouse models
Elena Shekhovtsova, Elena Svirshchevskaya and Marina Shevchenko
Immunology, Shemyakin & Ovchinnikov Institute of Bioorganic chemistry, Mikluho-Maklaya,
16/10, Moscow 117997, Russian Federation, Phone: +7 (495) 330-40-11, FAX: +7 (495) 330-
40-11, e-mail: shehovcova_elena@mail.ru

Aspergillus fumigatus (Af) fungi were shown to be potent to induce, in relation to immune sys-
tem status, Th1 or Th2 immune response. Thus, inhaling of Af conidia by patients under
immu-nosuppression leads to manifestation of invasive aspergillosis with Th1 type reaction,
when pa-tients, suffering from allergic asthma, can be at risk of allergic broncho-pulmonary
aspergillosis. The aim of the present study is to compare antibody subclasses of humoral
response in mouse models of invasive and allergic aspergillosis. Allergic aspergillosis was
induced by means of multiple injection of Asp f 2 – the major aller-genic protein from Af.
Antigen was supplied to mice in doses of 100 &#61549;g, 100 ng and 100 pg/mouse/injection,
i.p., 5 days a week during 2 weeks, in phosphate solution. Murine model of invasive
aspergillosis include inhalation of Af conidia followed by the course of i.p. injection of
cyclophosphamide (Cy) and cortisone acetate. Different doses from 0 to 3 mg/mouse/time of
Cy were used for the different groups of mice. The Asp f 2 specific serum antibody levels were
es-timated by ELISA. In mice with allergy to Af IgG1 was the most representative
immunoglobulin subclass. Serum IgA level decreased together with reduction of injected Asp f
2 dose. Opposite Asp f 2 specific IgE level was detected only in case of nanogramm dose
immunization. In mice with invasive as-pergillosis antibody secreting was significantly
reduced upon immunosuppression, the same time humoral response was represented with
IgG2a and IgA. The IgA level was reconstituted to the base level after the immunosuppression
cancellation and was reliably increased in group of mice, received 2 mg/mouse Cy. Under the
same conditions IgG level was significantly lower in this group. Consequently, Af invasion in
immunosuppressed mice drive the immune response to the Th1 type with IgG2a production.
The same time, immunocompetent mice show Th2 response with IgG1 and IgE production,
when sensitized in advance.




                                              191
P138C
Fungal infections following haploidentical hematopoietic transplantation associates to
polymorphisms in toll-like receptors
Agostinho Carvalho1, Lucia Pitzurra2, Teresa Aloisi2, Alessandra Carotti2, Patrícia Maciel1,
Franco Aversa2, António G. Castro1, Luigina Romani2 and Fernando Rodrigues1
1
  Infectious Diseases, Life and Health Sciences Research Institute (ICVS), Campus de Gualtar,
Braga      4710-057,     Portugal,    Phone: +351253604844,       FAX: +351253604831,      e-
mail: agostinho@ecsaude.uminho.pt 2 Dept of Experimental Medicine, Section of
Microbiology, University of Perugia, Perugia, Italy

The innate immune system is able to recognize conserved motifs of pathogens throughout
pattern recognition receptors, including Toll-like receptors (TLRs). Cellular activation via
TLRs triggers not only innate immune responses but also influences adaptive immunity.
Therefore, due to the significance of TLRs in the immune system, genetic variations within
these genes could have a major impact on the host immune response to pathogens. Fungal
infections are amongst the most significant causes of morbidity and mortality following bone-
marrow transplantation. Thus, we performed genetic association case-control studies to
evaluate a possible link between TLR polymorphisms, namely TLR2 (Arg677Trp and
Arg753Gln), TLR4 (Asp299Gly and Thr399Ile) and TLR9 (T-1923C, T-1486C, T-1237C,
G1174A and G2848A) and susceptibility to fungal infection in haploidentical hematopoietic
transplanted patients. In this study, 70 BMT patients, both with and without suspected fungal
infection were assessed for presence of the above-mentioned TLR polymorphisms. Our results
suggest that there is an association between the former TLR4 polymorphisms and susceptibility
to fungal infection, when considering patients with a clinical diagnosis of probable, possible or
proven fungal infection [20.1% vs. 6.9%, p=0.039]. This association can also be seen when
considering fungal colonization. Intriguingly, this association seems to be more frequent in
patients infected with yeast [31.0% vs. 8.5%, p=0.032] rather than with mould colonization.
This degree of association was even higher when considering only yeast colonization by others
than Candida albicans [48.3% vs. 6.9% p=0.029]. Further understanding of the basic molecular
mechanisms associated with susceptibility to fungal infections holds the promise of improving
diagnosis and therapeutic strategies to treat fungal infections particularly those associated with
hematopoietic transplantation.




                                                192
P139A
The PD1/PDL costimulatory pathway has a key regulatory role in Histoplasma
capsulatum pathogenesis
Attila Gacser1, Eszter Lazar-Molnar2, Steven C. Almo3, Stanley G. Nathenson4,
Gordon Freeman5 and Joshua D. Nosanchuk6
1
  Department of Medicine, Infectious Diseases, Albert Einsten College of Medicine (AECOM),
1300 Morris Park Ave, New York NY 10461, USA, Phone: +1 718 430 2993, FAX: +1 718
430 8968, e-mail: gacsera@gmail.com 2 Department of Cell Biology, AECOM 3 Department
of Biochemistry, AECOM 4 Department of Microbiology and Immunology, Cell Biology,
AECOM 5 Department of Medical Oncology, Dana Farber Canser Institute, Harvard Medical
School 44 Binney Street Boston, MA 02115 USA 6 Department of Medicine, Microbiology
and Immunology, AECOM

Costimulatory molecules such as members of the CD28/B7 family can either enhance or
attenuate T cell responses. The PD1 pathway negatively regulates T cell receptor signaling
upon interacting with its two ligands, PDL1 and PDL2, that are expressed on antigen
presenting cells (APCs). It is well established that the PD1/PDL system has a crucial role in
maintaining self-tolerance. However, pathogens can co-opt this pathway to escape immune
responses. In the present study, we examined the role of the costimulatory PD1/PDL pathway
in Histoplasma capsulatum (Hc) infection. Hc is an important human pathogenic dimorphic
fungus that primarily exists within macrophages in vivo. To characterize populations of
macrophages in murine histoplasmosis, peritoneal and alveolar macrophages were isolated and
analyzed at different time points after intranasal infection with Hc. qRT-PCR and FACS
analysis of these macrophages showed upregulation of PDL1. Splenocytes isolated form Hc
infected mice were significant upregulated as well, whereas PDL2 and PD1 levels were
unchanged. To investigate the mechanism of PDL1 upregulation in histoplasmosis, we infected
IFN-gamma -/- mice and analyzed the macrophages and splenocytes by FACS. Interestingly,
there was no upregulation of PDL1 on cells recovered from Hc infected IFN-gamma deficient
mice, suggesting that the upregulation of PDL1 is IFN-gamma-mediated. To further study the
importance of the PD1/PDL pathway in Hc infection, we used a PD1 deficient mouse model.
We have found that PD1-/- mice on the C57BL/6 background were completely resistant to
lethal Hc infection. Although they initially developed pulmonary inflammation, they were
disease free by day 10, as shown by histological analysis, and the pathogen could no longer be
cultured from liver, spleen or kidney. To investigate the therapeutical value of blocking the
PD1/PDL pathway, we treated Hc infected mice with a monoclonal antibody to PD1. Seventy
percent of the mice treated survived challenge with Hc, whereas all control mice (isotype
IgG2b and saline) died (p<0.001). Our results are the first to show the importance of the PD1
pathway in anti-fungal immunity and demonstrate that upregulation of PDL1 by Hc can
potently affect the interplay between host and pathogen. Moreover, our studies offer novel
potential immunotherapeutical strategies for histoplasmosis.




                                              193
P140B
Malassezia sympodialis enhances the mast cell IgE response and modifies the IL-6
production in a TLR-2 dependent manner
Christine Selander1, Gunnar Nilsson2, Annika Scheynius1 and Carolina Lunderius2
1
  Department of Medicine, Clinical Allergy Research Unit, Karolinska Institutet, Karolinska
University Hospital Solna, Stockholm 171 76, Sweden, Phone: +46 (0)8 517 76697, FAX: +46
(0)8 33 57 24, e-mail: Christine.Selander@ki.se 2 Department of Medicine, Clinical
Immunology and Allergy unit, Karolinska Institutet, Karolinska University Hospital Solna,
17176 Stockholm, Sweden

Atopic eczema (AE) is a chronic inflammatory puritic skin disorder, where the pathogenesis is
not fully understood. Factors contributing to the symptoms are genetic predisposition,
defective skin barrier and environmental factors such as microorganisms. Elevated serum
levels of IgE are frequent in AE patients, suggesting that allergens play a role in the pathogenic
mechanisms. The yeast Malassezia, part of the normal cutaneous flora, elucidates specific IgE-
and T-cell reactivity in AE patients. The genus comprises today of eleven species and M.
sympodialis is among the species most frequently isolated from both AE patients and healthy
controls. In AE the number of mast cells is increased in the skin. Mast cells are key effector
cells in IgE-associated Th2-like immune responses and have been shown to use Toll-like
receptors (TLRs) to respond to products of bacteria. We hypothesize that due to the ruptured
skin barrier in AE it is likely to believe that Malassezia can come in contact with mast cells
and direct the IgE activated mast cell towards an innate immune response through TLR
signalling. Bone marrow derived mast cells (BMMC) were generated from wild type (Wt) and
TLR-2 knock out (-/-) mice. The mast cells were incubated with trinitrophenyl (TNP) specific
IgE over night and then activated with TNP along with increasing amounts of M. sympodialis
extract. The cell supernatants were analysed after 0.5 h for mast cell degranulation by the beta-
hexosaminidase release assay and after 24 h for production of the pro-inflammatory cytokine
IL-6 by ELISA. M. sympodialis extract was found to enhance the IgE-mediated release of the
degranulation protein beta-hexosaminidase by BMMC from Wt and TLR-2 -/- mice.
Furthermore, M. sympodialis extract modified the IL-6 production of IgE activated mast cells
from Wt mice in a dose dependent manner, where addition of low concentrations of M.
sympodialis extract led to an increase in the IL-6 production and high concentrations to a
decrease. However, no effect of M. sympodialis extract was seen on the IL-6 production using
BMMC derived from TLR-2 -/- mice, indicating the dependence on signalling through the
TLR-2 receptor. In conclusion M. sympodialis enhances the mast cell IgE response and alters
the IL-6 production in a dose dependent manner by signalling through the TLR-2 receptor.
These effects of M. sympodialis on IgE activated mast cells could contribute to the
inflammation in AE.




                                                194
P141C
Candida spp activate interferon-type I response in mouse myeloid dendritic cells.
Christelle Bourgeois1, Olivia Majer1, Ingrid Frohner1, Silvia Stockinger2, Thomas Decker2
and Karl Kuchler1
1
  Department of Medical Biochemistry, Medical University of Vienna, Dr. Bohr-gasse 9/
Ebene2, Vienna A-1030, Austria, Phone: + 43 1 4277-61811, FAX: + 43 1 4277-9618, e-
mail: christelle.bourgeois@meduniwien.ac.at 2 University of Vienna

Harmless commensals in most healthy people, Candida spp can cause both superficial
infections and life-threatening systemic candidiasis in immunocompromised patients. The most
frequently encountered species in humans are Candida glabrata (Cg), which exists only in
yeast form, and two dimorphic species Candida albicans (Ca) and Candida dubliniensis (Cd),
both of which share the ability to filament. The dimorphic switch from yeast to hyphae is
considered a major virulence determinant, and also implicated in evading the host immune
system. Cells of the innate immune system (e.g, dendritic cells, macrophages) comprise the
first line of defense against pathogens and largely determine the outcome of infection. They are
major producers of interferon (IFN) beta, a type I IFN. While IFNs may also play important
roles in combating bacterial infections, their role in defense against fungal pathogens is as yet
unknown. Hence, our work aimed to address a possible type I INF response in mouse myeloid
dendritic cells (mDCs) responding to Candida challenges in vitro. Our results indicate that Cg,
as well as the dimorphic species Ca and Cd, trigger production of INF-beta in mDCs. This
stimulation is independent from hyphae formation since Cg only exists in the yeast form.
Induction of IFN-beta transcription also correlated with activation of STAT1 at Tyr701. UV-
treated Ca stimulated STAT1 activation as well, although to a lesser extent than live fungal
cells. Thus, all Candida spp tested are able to induce a type I IFN response in mDCs,
suggesting that the IFN-beta response may play an important role in the defense against fungal
infections.




                                                195
P142A
Interactions between Histoplasma capsulatum and host macrophages
Charlotte Berkes, Diane Inglis and Anita Sil
Department of Microbiology and Immunology, University of California, San Francisco, 513
Parnassus Avenue 0414, San Francisco CA 94143, USA, Phone: 00 1 415-502-4810, FAX: 00
1 415-476-8201, e-mail: Charlotte.Berkes@ucsf.edu

Histoplasma capsulatum is a dimorphic fungal pathogen that infects humans, causing
respiratory and systemic disease in immunocompromised hosts. H. capsulatum spores, or
conidia, are delivered to the host via the respiratory route and are initially phagocytosed by
alveolar macrophages. In vivo conditions promote conidial germination and formation of
yeasts, which replicate within the macrophage phagosome, establishing long-term infection
within the host. In order to understand the mechanisms by which H. capsulatum yeast and
conidia interact with host macrophages, we have developed an in vitro infection model using
bone marrow-derived macrophages (BMDMs). While infection of BMDMs with either
morphological form of H. capsulatum results in macrophage lysis, the kinetics of cell death
differ -- infection with yeasts results in rapid lysis, whereas conidia-induced lysis is delayed by
several days. Transcriptional profiling of macrophage gene expression following infection with
either the yeast or conidial form of H. capsulatum highlights genes that are induced in response
to both yeast and conidia, as well as genes induced specifically upon infection with conidia or
yeasts. Both forms of H. capulatum cause induction of proinflammatory cytokines such as TNF
alpha, as well as genes known to regulate apoptosis. However, infection of macrophages with
conidia, but not yeasts, results in expression of interferon response genes, highlighting a
potentially critical difference in the host response to different morphologies of H. capsulatum.
Current experiments are aimed at understanding the signaling pathways responsible for
orchestrating the innate immune response to this important respiratory pathogen.




                                                 196
P143B
Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms
with a defined set of antimicrobial proteins.
Constantin Urban1, David Ermert1, Monika Schmid2, Ulrike Abed3, Volker Brinkmann3,
Peter Jungblut2 and Arturo Zychlinsky1
1
  Cellular Microbiology, Max Planck Institute for Infection Biology, Charitéplatz 1, Berlin
10117, Germany, Phone: +49 (0)30 28460 357, FAX: +49 (0)30 28460 301, e-
mail: curban@mpiib-berlin.mpg.de, Web: http://www.mpiib-berlin.mpg.de/ 2 Core facility for
Protein Analysis, Max Planck Institute for Infection Biology, Charitéplatz 1, 10117 Berlin,
Germany 3 Core facility for Microscopy, Max Planck Institute for Infection Biology,
Charitéplatz 1, 10117 Berlin, Germany

Candida albicans is the predominant etiologic agent of fungal infections in humans.
Neutrophils are essential in controlling candidiasis. It is well established that neutrophils
phagocytose and kill C. albicans upon phagolysosomal fusion. Recently we found that
activated neutrophils release granule proteins and chromatin that together form extracellular
fibers, called Neutrophil Extracellular Traps (NETs). We show that C. albicans stimulates
neutrophils to form NETs in vitro. Moreover the NETs trap and kill both yeast-form and
hyphal cells which we demonstrate in time lapse movies. To understand how NETs kill C.
albicans on the molecular level we are analysing the NET-proteome. We isolate the released
NETs, and identify the associated proteins by mass spectrometry. So far this approach resulted
in the identification of approximately 20 different proteins. Using immunohistochemistry we
confirmed their localisation to NETs. We are now assessing the contribution of the identified
NET-proteins to the killing of C. albicans. Furthermore we are localizing the NET-proteins in
experimental candidiasis. Taken together our data will give insight into how neutrophils
contribute to the clearance of fungal infections.




                                              197
P144C
Predicting substrates of fungal Kex2 proteinases on genomic scale
Oliver Bader1 and Bernhard Hube2
1
  Medical Microbiology, Georg-August-University Göttingen, Kreuzbergring 57, Göttingen
37075, Germany, Phone: +49 (0)551 39 2346, FAX: +49 (0)551 39 5861, e-
mail: obader@gwdg.de 2 Hans Knoell Institute Jena (HKI), Beutenbergstraße 11a, 07745 Jena,
Germany

The Kex2 proteinase dependant processing system is highly conserved between different fungi.
This Golgi-resident system is essential for the proteolytic activation of mating pheromones and
several other proteins destined for the extracellular space or the cell wall, such as adhesins or
enzymes. Earlier studies have identified subtrates of fungal Kex2 proteinases, that are not
conform to the established substrate specificity models for these proteinases, e.g. the
Saccharomyces cerevisiae killertoxin precursor. Here, we describe how this model can be
improved by 3D-modelling of interactions of substrates with proteinases and how structural
features of substrates are associated with proteolytic cleavage. An algorithm was developed to
predict Kex2 substrate sites on a whole-genome basis and the predictions were confirmed by in
vitro testing of selected heterologously expressed substrate proteins with recombinant
proteinases. In addition to known or predicted substrates of Kex2, we identified several new of
the human pathogenic fungus Candida albicans, including OP4-homologous proteins and the
most abundant hyphae-specific protein Ece1. Even though the Kex2 proteinases of C. albicans,
C. glabrata, Pichia pastoris and Saccharomyces cerevisiae are extremely homologous, we
were able to show distinct differences in substrate recognition in the recombinantly produced
enzymes for at least one substrate. The identified substrates were then used to explain the
pleiotropic phenotype of the deletion mutants.




                                                198
P145A
Decoding serological response to Candida cell wall immunome into novel diagnostic,
prognostic, and therapeutic candidates for systemic candidiasis by proteomic and
bioinformatic analyses.
Aida Pitarch1, Antonio Jiménez2, César Nombela1 and Concha Gil1
1
  Microbiology II, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y
Cajal, s/n, Madrid 28040, SPAIN, Phone: +34 91 394 1755, FAX: +34 91 394 1745, e-
mail: apitavel@farm.ucm.es, Web: http://www.ucm.es/ 2 Department of Internal Medicine II,
Salamanca University Hospital, 37007 Salamanca, Spain

In an effort to bring novel diagnostic and prognostic biomarkers or even potential targets for
vaccine design for systemic candidiasis (SC) into the open, a systematic proteomic approach
coupled with bioinformatic analysis was used to decode the serological response to Candida
wall immunome in SC patients. Serum levels of IgG antibodies against Candida wall-
associated proteins (proteins secreted from protoplasts in active wall regeneration, separated by
two-dimensional gel electrophoresis, and identified by mass spectrometry) were measured in
45 SC patients, 57 non-SC patients, and 61 healthy subjects by Western blotting. Two-way
hierarchical clustering and principal component analysis of their serum anti-Candida wall
antibody expression patterns discriminated SC patients from controls and highlighted the
heterogeneity of their expression profiles. Multivariate logistic regression models demonstrated
that high levels of antibodies against glucan 1,3-beta-glucosidase (Bgl2p) and the anti-wall
phosphoglycerate kinase antibody seropositivity were the only independent predictors of SC.
Receiver operating characteristic curve analysis revealed no difference between their combined
evaluation and measurement of anti-Bgl2p antibodies alone. In a logistic regression model
adjusted for known prognostic factors for mortality, SC patients with high anti-Bgl2p antibody
levels or a positive anti-wall enolase antibody status, which correlated with each other, had a
reduced 2-month risk of death. After controlling for each other, only the seropositivity for anti-
wall enolase antibodies was an independent predictor of a lower risk of fatality, supporting that
these mediated the protective effect. No association between serum anti-cytoplasmic enolase
antibody levels and outcomes was established, suggesting a specific mechanism of enolase
processing during wall biogenesis. We conclude that serum anti-Bgl2p antibodies are a novel
accurate diagnostic biomarker for SC and that, at high levels, they may provide protection by
modulating the anti-wall enolase antibody response. Furthermore serum anti-wall enolase
antibodies are a new prognostic indicator for SC and confer protection against it. Bgl2p and
wall-associated enolase could be valuable candidates for future vaccine development.




                                                199
P146B
Candida spp. trigger ROS production in primary macrophages & dendritic cells
Ingrid Frohner, Christelle Bourgeois, Olivia Majer and Karl Kuchler
Medical Biochemistry, Medical University, Max F. Perutz Laboratories, Dr. Bohrgasse 9/2,
Vienna A 1020, Austria, Phone: 0043 1 4277 61818, FAX: 0043 1 4277 9618, e-
mail: ingrid.frohner@meduniwien.ac.at

The clinical spectrum of the human commensals such as Candida spp. ranges from
mucocutaneous infections to systemic life-threatening diseases in immunocompromised
patients. Monocytes are considered the first defence line combating these pathogens. One of
the immediate early response of macrophages and dendritic cells phagocytosing fungi is the
production of reactive oxygen species (ROS), which play important roles in the modulation of
inflammatory reactions. ROS destroy invading pathogens, but overproduction of ROS may
also have detrimental effects, as it also causes endothelial damage. Previous studies have
shown that zymosan, a cell wall preparation of Saccharomyces cerevisiae, as well as the C.
albicans (C.a) in the yeast form, strongly induce ROS in macrophages. Thus, we asked whether
Candida. spp can induce ROS in primary bone marrow-derived macrophages (BMDM), as well
as in myeloid dendritic cells (mDC), as these cell types represent key factors in the defense
against fungal infections. Using in vitro ROS assays based on luminol-enhanced
chemiluminescence as well as dihydrorhodamine (DHR) staining, we showed that C.g, C.
dubliniensis (C.d), and C.a induce ROS in BMDMs as well as in mDCs, although to a different
extent and with different kinetics. The ROS response increased up to a multiplicity of infection
of 10:1 for C.a, but decreased at higher MOIs until it was completely eliminated at a MOI of
50:1. Interestingly, C.d induced ROS much stronger than C.a, whereas C.g was a very weak
ROS inducer. Laminarin inhibited 70-80% of the ROS production in mDCs, but only 30-50%
in BMDM, indicating that dectin-1 may not the only receptor involved in Candida-stimulated
ROS production in BMDMs. Preliminary results showed that blocking phagocytosis by
Cytochalasin D dramatically increased zymosan-induced ROS signalling, but blocked
Candida-triggered ROS signalling. These results suggest that adhesion-dependent extracellular
and phagocytosis-dependent intracellular pathways are involved in triggering ROS production.
The marked differences in ROS production in BMDMs and mDCs infected by C.a, C.d and
C.g may relate to differences in virulence observed for these fungal pathogens in vivo.




                                               200
P147C
Analysis of the transcriptional response of mouse macrophages to infection with Candida
glabrata.
John Synnott, Mary E Logue, Claire Kenny and Geraldine Butler
UCD School of Biomolecular and Biomedical Science, Conway Institute, University College
Dublin, Belfield, Dublin Dublin 4, Ireland, Phone: +353-(0)1-176-6838, FAX: +353-(0)1-283-
7211, e-mail: john.synnott@ucd.ie, Web: http://www.ucd.ie/conway/cv_72.html

Candida glabrata is an opportunistic pathogenic fungus, and accounts for a high percentage of
candidiasis. Systemic and mucosal infections are common among immunocompromised
patients such as individuals infected with AIDS or diabetes mellitus. Little is known about the
virulence of C. glabrata or the regulation of genes involved in the host response. The
transcription factor Ace2 from C. glabrata controls the expression of many cell wall genes. An
ace2 knockout strain has a clumpy phenotype and is hypervirulent in a mouse model. In an
attempt to understand this hypervirulent phenotype, we examined the gene expression in RAW
264.7 murine macrophage cells infected with wild type C. glabrata cells and ace2 knock out
cells. Cytoplasmic RNA was extracted from macrophages at 0, 1, 2, and 4 h following
infection. The labelled cDNA was combined with cDNA from uninfected macrophage cells
and hybridised to Agilent Mouse Whole Genome 44k Arrays.Quality control and Lowess
normalisation were carried out in GeneSpring. Initial statistical analysis identified 512 genes
with significant alterations in expression. Many genes involved in apoptosis were highly up-
regulated in macrophages infected with both wild type and ace2 knock out cells. However the
increases were higher in response to infection with wild type cells. The genes include
SQSTM1, TRIB3, GADD45B, DEDD2, BCAR3, LITAF, TRAF1, AXUD1, and MCL1.
Expression of genes involved in cytoskeletal organisation such as EPS8, ARL8, RHOQ, and
PDGFA were up-regulated equally in both infection series. Genes encoding proinflammatory
chemokines, including CXCL2, CCL3 (Macrophage Inflammatory Protein- alpha), CCL4
(MIP-1beta), and CCL7 were also up-regulated at 1 to 4 h following infection. In addition,
genes involved in activation of the I-kappaB kinase/NF-kappaB cascade (the TRAF-binding
protein TANK, and the proinflammatory cytokine TNF-alpha), were up-regulated. Conversely,
the genes KPNA2, PURL3, CLEC7A, and KIFAP3 involved in cell-cell adhesion were down-
regulated, as was the gene TXNIP which functions in response to oxidative stress. These results
provide valuable insights into the mechanisms of C. glabrata pathogenesis and the host-
pathogen immune response. Initial analysis suggests that the ace2 knock out strain is less likely
to induce apoptosis, which may be related to its hyper-virulent phenotype.




                                                201
P148A
Comparison of methods for the isolation and identification of Candida dubliniensis
Michael Fleischhacker, Julia Pasligh, Grzegorz Kofla, Clarissa Radecke and Markus Ruhnke
Med. Klinik m.S. Onkologie/Hämatologie, Med. Charité-Universitätsmedizin Berlin,
Charitéplatz 1, Berlin 10117, Germany, Phone: +4930 450 51 33 04, FAX: +4930 450 51 39
64, e-mail: michael.fleischhacker@charite.de

Many of the fast and cheap methods wich are in use for the isolation, characterization and
discrimination of different Candida species lack sensitivity and/or specificity. In contrast
molecular genetic assays demonstrate a higher sensitivity and specifity, but are more labor
intensive and more expensive. Frequently C. dubliniensis is not found as a single
microorganism, but in association with other Candida species when isolated from an oral
cavity. But there are also reports which demonstrated that C. dubliniensis is the sole infectious
organism isolated from symptomatic and asymptomatic HIV-positive patients, suggesting that
C. dubliniensis might be pathogenic at least in HIV patients. The increased clinical relevance
of C. dubliniensis infections and an unambiguous identification of this organism is still a
problem. The aim of our work was to compare several established cultural methods for the
identification of Candida dubliniensis. We used a panel of different cultivation methods for the
characterization of 199 clinical isolates obtained from 122 samples which werr taken from the
oral cavity. The results obtained with the cultivation methods were compared with the AP-PCR
which was considered the gold standard for these experiments. We achieved a sensitivity of 14
% for the identification on CHROMagar®, 42 % for the identification on rice agar, 100 % for
discrimination on bird-seed agar, 100 % with an assimilation profile index (API ID 32C) and
97 % when grown at increased temperature, i.e. 45°C. Most of our data are in good accordance
with the API ID 32 C reference manual. The most striking exception is the assimilation of
palatinose in 88 % of our C. dubliniensis isolates in contrast to 0 % in the API reference
manual. From our experiments we conclude that a panel of different identification methods is
necessary to achieve a high level of sensitivity and specficity for the identification of C.
dubliniensis. According to these results we propose an optimized flow chart for a clear-cut
discrimination of C. dubliniensis from C. albicans.




                                                202
P149B
Host/Candida interactions at mucosal surfaces
David Moyes1, Qianfan Bai1, Guenther Weindl2, Martin Schaller2 and Julian Naglik1
1
  Oral Immunology, King's College London, St Thomas Street, London SE1 9RT, UNITED
KINGDOM, Phone: +44 20 7188 4377, FAX: +44 20 7188 4375, e-
mail: julian.naglik@kcl.ac.uk 2 Department of Dermatology, Eberhard Karls University
Tübingen, Tübingen, Germany

The mucosal epithelium has immense importance in host defense and immune surveillance, as
it is the cell layer that initially encounters most microorganisms. Immune responsiveness to
many microbes depends on a family of innate recognition molecules known as pattern
recognition receptors (PRR’s), which are the major innate recognition system for microbial
invaders in eukaryotes. They are triggered by conserved molecular structures (pathogen-
associated molecular patterns (PAMPs)) expressed by bacteria, viruses and fungi. The three
main PRRs known to respond to Candida albicans in myeloid cells are (toll-like receptor)-2,
TLR4, and dectin-1/hbGR, which recognize phospholipomannans, mannans, and b-glucans.
However, the C. albicans cell wall components that specifically induce epithelial PRR
responses have not yet been investigated in detail. In this study, using an established model of
oral candidosis based on reconstituted human oral epithelium (RHE), we examined the
responsiveness of oral epithelium to TLR1-9 agonists, different Candida species, and C.
albicans cell wall mutants defective in N- and O-mannosylation. All TLR1-9 agonists
stimulated IL-6 (especially poly (I:C) (TLR3) and imiquimod (TLR7/8)), G-CSF, TNFa, and
GM-CSF (except ODN2006), demonstrating their presence and activity. IL-8 is constitutively
secreted at high levels, but is further induced by most TLR agonists. Infection with wild type
C. albicans (SC5314), but not other Candida species, induced tissue damage (measured by
lactate dehydrogenase release) and a cytokine response (IL-1b, IL-6, TNFa, IL-8, and GM-
CSF). However, TLR1-10 upregulation was not clearly evident by real-time PCR. When
examining four C. albicans cell wall mutants, only och1 (severe N-mannosylation mutant) was
attenuated in its ability to cause tissue damage in the oral RHE. och1 was also less able to
induce IL-1b, IL-6, TNFa and GM-GSF. Of the other C. albicans cell wall mutants, pmr1 (N-
and O-mannosylation mutant) possessed reduced ability to induce TNFa, the mnt1/2 mutant
(O-mannosylation) was attenuated in inducing IL-6, and mnn4 (N-mannosylation mutant) had
no observed phenotype in tissue damage or cytokine induction compared with SC5314. We
have demonstrated presence and activity of TLR1-9 in epithelium. Also, the RHE model is an
ideal tool to characterise host/pathogen interactions and to study the mechanisms involved in
the PRR-mediated detection of C. albicans at mucosal surfaces.




                                               203
P150C
Immune response to Candida albicans is preserved despite defect in O-mannosylation of
secretory proteins
Franck      Alexandre Skrzypek1,      Cristina Corbucci1,  Elio Cenci1,    Elena Gabrielli1,
             1             2                    1                     1
Paolo Mosci , Joachim Ernst , Francesco Bistoni and Anna Vecchiarelli
1
  Experimental Medicine and Biochemical Sciences, University of Perugia, Via Del Giochetto,
Perugia 06126, ITALY, Phone: +39 (0)75 / 5857407, FAX: +39 (0)75 / 5857407, e-
mail: frafabs@hotmail.com 2 Institut fur Mikrobiologie, Heinrich-Heine-Universitat 40225
Dusseldorf Germany

The PMT gene family in Candida albicans encodes five isoforms of protein
mannosyltransferases that initiate O-mannosylation of secretory proteins. Mutations at Pmt
level have been associated with different pathogenicities. In particular, differently from
pmt5/pmt5, pmt2/PMT2 revealed poor virulence. We intended to determine whether the
different grade of pathogenicity is related to the capacity of pmt2/PMT2 and pmt5/pmt5 to: i)
express differences in selected virulence factors; ii) stimulate the natural immune system. The
results show that pmt mutants: i) form hyphae in serum; ii)show defective production of
proteases but not of phospholipases with respect to the parental strain; iii) undergo mycelial
transition in the kidneys of hematogenously infected animals; iv) are phagocytosed and killed
by macrophages similarly to the parental strain, although neutrophils are unable to destroy
pmt5/pmt5; v) engage TLR4 and stimulate MyD88 leading to NF-kB activation; vi) stimulate
cytokine production by macrophages. Collectively our findings suggest that the defect in
protein O-mannosylation in Candida albicans implies attenuation of the virulence although the
antigenic equipment that retains the capacity to stimulate an efficient immune response is
preserved. Thus, the low or high virulence of strains does not necessarily correlate with good
or bad immune response.




                                               204
P151A
Generation of recombinant antibodies against major allergens of Aspergillus fumigatus
Mark Schütte, Martina Kirsch, Sonja Eichholz, Dominik Hinz, Thomas Schirrmann,
Michael Hust and Stefan Dübel
Department of biotechnology and biochemistry, TU Braunschweig, Spielmannstrasse 7,
Braunschweig 38106, Germany, Phone: +49 (0) 53 13 91 57 60, FAX: +49.531.391.5763, e-
mail: ma.schuette@tu-bs.de, Web: www.tu-braunschweig.de/bbt

The opportunistic fungus Aspergillus fumigatus is the etiological agent of invasive
aspergillosis in immunosuppressed or immunocompromised patients. However, there is still an
unmet yet urgent need for diagnostic methods concerning improved reliability, sensitivity and
early diagnosis. In this work we isolated four genes encoding major Aspergillus fumigatus
allergens. The isolated genes encode peroxisomal membrane protein (PMP, Asp f3),
manganese superoxide dismutase (MnSOD, Asp f6), a metalloprotease (MEP, Asp f5) and a
new gene related to glycosyl hydrolase (Crf) named Crf2. All corresponding proteins were
produced in E. coli BLR(DE3) using the pET21A + vector system. After His-tag and
subsequent ion exchange chromatography, these proteins were used as targets for the isolation
of single chain fragments variable (scFv) using antibody phage display. Eleven scFvs against
PMP, eight against MnSOD and seven directed against the MEP were isolated and
biochemically characterised. The selection of antibody fragments specific for Crf2 is in
progress. The antibody fragments will be further characterised concerning their ability to detect
Aspergillus fumigatus antigens in serum or lavage by sandwich ELISA. Additionally, the
scFvs will be checked for their applicability for passive immunisation in order to prevent
Aspergillus fumigatus infections.




                                                205
P152B
Physiological effects of pacC silencing on Aspergillus flavus
Essa Suleman and Benesh M. Somai
Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O Box 77000,
University Way, Summerstrand, Port Elizabeth 6000, South Africa, Phone: +27 (0)41 504
2608, FAX: +27 (0)41 504 2814, e-mail: Essa.Suleman@nmmu.ac.za

Many microorganisms, and in particular fungi, are able to grow over a wide pH range. These
microorganisms must possess some regulatory system that senses environmental pH and
ensures that gene expression is tailored to the pH of the environment. In Aspergillus pH
regulation is mediated by palA, palB, palC, palF, palH, palI and pacC genes. The activated
form of the PacC protein activates genes that are required at alkaline pH, e.g. genes coding for
alkaline phosphatases, and represses certain genes that are functional at acidic pH, e.g. genes
encoding acid phosphatases. PacC also positively regulates genes involved in penicillin
biosynthesis, e.g. the isopenicillin N synthase gene, ipnA, in A. nidulans. It has been shown
that pacC, in Fusarium oxysporum acts as a negative virulence regulator in plants and it was
hypothesised that PacC prevents expression of genes that are important for infection and
virulence of the pathogen (Caracuel, Z, et al, (2003), Mol Microbiol, Volume 8, p765). To
elucidate the physiological effects that pacC had on growth, conidiation and the pathogenicity
of A. flavus, a novel RNA interference method was utilized. This involved cloning of a partial
pacC gene fragment in the forward and reverse orientations in a fungal expression cassette to
create an RNA interference (RNAi) vector. The unique structure of this vector allowed the
cloned fragments to be expressed forming a double stranded stem-loop structure or short
hairpin RNA (shRNA). Formation of shRNA resulted in activation of the endogenous RNA
interference pathway and subsequent silencing of pacC. The results showed that pacC did not
play a significant role in primary growth and development but adversely affected production
and morphology of conidia at alkaline pH. Furthermore, pacC RNAi silencing severely
impaired the ability of the A. flavus mutants to infect and cause damage of maize. Scanning
electron microscopy indicated that pathogenicity of A. flavus on maize is directly related to the
structural integrity of conidia, which in turn is greatly influenced by pacC. This study showed
that pacC positively regulates expression of one or more genes involved in conidiation and
pathogenicity in A. flavus under alkaline conditions.




                                                206
LIST OF PARTICIPANTS

        AND

 INDEX OF AUTHORS




         207
                                     List of Participants

Laura ALCAZAR FUOLI                                    Lilian Cristiane BAEZA
Servicio Micología                                     Departamento de An·lises ClÌnicas
Centro Nacional Microbiología, CNM                     UNESP - Universidade Estadual Paulista
Ctr. Pozuelo-Majadahonda Km 2                          Rua Expedicion·rios do Brasil, 1621
Madrid sp 28220, spain                                 Araraquara SP 14801-902, Brazil
Tel : 34918223661                                      Tel : +55 (16) 3301-6556
Fax : 34915097034                                      Fax : +55 (16) 3301-6547
Mail: lalcazar@isciii.es                               Mail: baezalc@fcfar.unesp.br
Web : www.isciii.es
                                                       Martine BASSILANA
Agostinho ALMEIDA                                      Inst. of Signaling, Developmental Biology &
Life and Health Sciences Research Institute            Cancer Research
(ICVS)                                                 CNRS UMR6543
University of Minho                                    FacultÈ des Science, Parc Valrose UNSA
Campus de Gualtar                                      Nice 6108, France
Braga 4710-057, Portugal                               Tel : +33 (0)4 076464
Tel : 351253604 844                                    Fax : +33 (0)4 076466
Fax : 351253604 831                                    Mail: mbassila@unice.fr
Mail: ajalmeida@ecsaude.uminho.pt
                                                       Robert J BASTIDAS
James B. ANDERSON                                      Department of Molecular Genetics and
Cell and Systems Biology                               Microbiology
University of Toronto                                  Duke University
3359 Mississauga Road North                            322 CARL Bldg., Research Drive, Box 3546
Mississauga ON L5L 1C6, Canada                         Durham NC 27710, USA
Tel : 905 828-5362                                     Tel : +001-919 684-2809
Fax : 905-828-3792                                     Fax : +001-919 684-5458
Mail: janderso@utm.utoronto.ca                         Mail: rjb10@duke.edu
                                                       Web :
Jorge ANJOS                                            http://mgm.duke.edu/faculty/heitman/index.ht
Centre for Neurosciences and Cell Biology              m
Institute of Microbiology, Faculty of Medicine
Universidade de Coimbra, Rua Larga                     Christopher BAUSER
Coimbra 3004-504, Portugal                             GATC Biotech
Tel : -239834378                                       Jakob-Stadler Platz 7
Fax : -239826447                                       Konstanz 78467, Germany
Mail: jorge.f.anjos@gmail.com                          Tel : +49 (0)173 8160 0
                                                       Fax : +49 (0)173 8160 81
Oliver BADER                                           Mail: c.bauser@gatc-biotech.com
Medical Microbiology                                   Web : http://www.gatc-biotech.com
Georg-August-University Gˆttingen
Kreuzbergring 57                                       Anne BEAUVAIS
Gˆttingen 37075, Germany                               Parasitologie-Mycologie
Tel : +49 (0)551 39 2346                               Institut Pasteur
Fax : +49 (0)551 39 5861                               25 rue du Docteur Roux
Mail: obader@gwdg.de                                   Paris 758015, France
                                                       Tel : 01 45 68 82 25
                                                       Fax : 01 40 61 34 19
                                                       Mail: abeauvai@pasteur.fr




                                                 208
Charlotte BERKES                                    Marianne BOLSTAD
Department of Microbiology and Immunology           Biosciences
University of California, San Francisco             University of Kent
513 Parnassus Avenue 0414                           Giles Lane
San Francisco CA 94143, USA                         Canterbury CT27NJ, United Kingdom
Tel : 00 1 415-502-4810                             Tel : +44 (0)1227 82 3735
Fax : 00 1 415-476-8201                             Fax : +44 (0)1227 76 3912
Mail: Charlotte.Berkes@ucsf.edu                     Mail: mb259@kent.ac.uk

Judith BERMAN                                       Marie-Elisabeth BOUGNOUX
Genetics, Cell Biology & Development                Fungal Biology and Pathogenicity
University of Minnesota                             Institut Pasteur
321 Church St. SE, 6-160 Jackson H                  25 rue du Docteur Roux
MInneapolis MN 55455, USA                           Paris 75015, FRANCE
Tel : 612-625-1971                                  Tel : +33 (0)1 45 68 86 19
Fax : 612-626-6140                                  Fax : +33 (0)1 4568 8938
Mail: jberman@umn.edu                               Mail: bougnoux@pasteur.fr
Web :
http://www.cbs.umn.edu/labs/berman/index.ht         Christelle BOURGEOIS
m                                                   Department of Medical Biochemistry
                                                    Medical University of Vienna
Jessica BESER                                       Dr. Bohr-gasse 9/ Ebene2
Parasitology, Mycology and Environmental            Vienna A-1030, Austria
Microbiology                                        Tel : + 43 1 4277-61811
Swedish Institute for Infectious Disease            Fax : + 43 1 4277-9618
Control                                             Mail: christelle.bourgeois@meduniwien.ac.at
Nobels v‰g 18
Solna 17182, Sweden                                 Martina BRACHHOLD
Tel : +46 (0)8 4572544                              Molecular Biotechnology
Fax : +46 (0)8 318450                               Fraunhofer Institute
Mail: jessica.beser@smi.ki.se                       Nobelstrasse 12
Web : www.smittskyddsinstitutet.se                  Stuttgart 70569, Germany
                                                    Tel : +49 (0)711 970 4145
Elaine BIGNELL                                      Fax : +49 (0)711 970 4200
Department of Molecular Microbiology and            Mail: Martina.Brachhold@igb.fhg.de
Infection                                           Web : www.igb.fraunhofer.de
Imperial College London
Armstrong Road                                      Alistair JP BROWN
London SW72AZ, United Kingdom                       School of Medical Sciences
Tel : 442075942 074                                 University of Aberdeen
Fax : 442075943 076                                 Institute of Medical Sciences, Foresterhill
Mail: e.bignell@imperial.ac.uk                      Aberdeen AB25 2ZD, United Kingdom
                                                    Tel : +44 (0)1224 555883
Arnold BITO                                         Fax : +44 (0)1224 555844
Department of Cell Biology                          Mail: al.brown@abdn.ac.uk
University of Salzburg                              Web :
Hellbrunnerstrasse 34                               www.abdn.ac.uk/ims/staff/details.php?id=al.br
Salzburg 5020, Austria                              own
Tel : 4366280445 793
Fax : 436628044 144
Mail: arnold.bito@sbg.ac.at




                                              209
Gordon BROWN                                            Julia CALDERON BLANCO
IIDMM                                                   Dipartimento di Scienze Biomolecolare e
University of Cape Twon                                 Biotecnologie
Anzio Road                                              Universit‡ di Milano
Cape Town 7925, South Africa                            Via Celoria 26
Tel : 27 21 406 6684                                    Milano It 20133, Italy
Fax : 27 21 406 6029                                    Tel : +39(0)25031 4920
Mail: gordon.brown@mweb.co.za                           Fax : +39(0)25031 4912
Web : http://www.iidmm.uct.ac.za/gbrown/                Mail: julia.calderon@unimi.it
                                                        Web : www.unimi.it
Sascha BRUNKE
Microbial Pathogenicity Mechanisms                      Pilar D. CANTERO
Hans Knoell Institut                                    Departamento de MicrobiologÌa y GenÈtica
Beutenbergstr. 11a                                      Universidad de Salamanca
Jena 7745, Germany                                      Campus Miguel de Unamuno
Tel : +49 (0)3641 65 6885                               Salamanca 37007, Spain
Fax : +49 (0)3641 65 6882                               Tel : 34923294 677
Mail: sascha.brunke@hki-jena.de                         Fax : 34923224 876
                                                        Mail: soile@usal.es
Helena BUJDAKOVA
Microbiology and Virology                               Nicole CAPLICE
Comenius University, Faculty of Natural                 Microbiology Research Unit, Division of Oral
Sciences                                                Biosciences
Mlynska dolina B-2                                      Dublin Dental School and Hospital
Bratislava SK 84215, Slovakia                           Lincoln place
Tel : 421260296446                                      Dublin D 2, Ireland
Fax : 421265429064                                      Tel : +353 (0)1 612 7350
Mail: bujdakova@fns.uniba.sk                            Fax : +353 (0)1 612 7295
Web : http://www.fns.uniba.sk/~bujdakova/               Mail: nicole.caplice@dental.tcd.ie

Geraldine BUTLER                                        Laura Elena CARRETO-BINAGHI
Conway Institute                                        Lab. InmunologÌa de Hongos,Dept.
University College Dublin                               MicrobiologÌa-ParasitologÌa
Belfield                                                Facultad Medicina, Universidad Nacional
Dublin 4, Ireland                                       AutÛnoma de MÈxico
Tel : -7166533                                          Universidad 3000, Circuito escolar, Ciudad
Fax : -2836859                                          Universitaria
Mail: geraldine.butler@ucd.ie                           Mexico 4510, Mexico
Web :                                                   Tel : +52 (55) 5623-2462
http://www.ucd.ie/sbbs/staff/butler_geraldine/i         Fax : +52 (55) 5623-2462
ndex.html                                               Mail: lauraelena_c@yahoo.com
                                                        Web : www.histoplas-mex.unam.mx
Virginia CABEZON
Microbiology II                                         Agostinho CARVALHO
Faculty of Pharmacy. UCM                                Infectious Diseases
Plaza RamÛn y Cajal s/n                                 Life and Health Sciences Research Institute
Madrid 28040, Spain                                     (ICVS)
Tel : 34913941755                                       Campus de Gualtar
Fax : 34913941745                                       Braga 4710-057, Portugal
Mail: vcabezon@farm.ucm.es                              Tel : 351253604844
Web : www.ucm.es                                        Fax : 351253604831
                                                        Mail: agostinho@ecsaude.uminho.pt




                                                  210
Arturo CASADEVALL                                   Moira COCKELL
Microbiology & Immunology                           Institute of Microbiology
Albert Einstein College of Medicine                 CHUV
1300 Morris Park Avenue                             Rue du Bugnon 48
Bronx NY 10461, USA                                 Lausanne VD CH-1011, Switzerland
Tel : 718-430-3665                                  Tel : +41 (0)21 314 4084
Fax : 718-430-8711                                  Fax : +41 (0)21 314 4060
Mail: casadeva@aecom.yu.edu                         Mail: moira.cockell@hospvd.ch
Web : www.aecom.com                                 Web :
                                                    http://www.chuv.ch/imul/imu_home/imu_rech
Antonio CASSONE                                     erche/imu_recherche_hauser.ht
Infectious Diseases
Istituto Superiore di Sanita                        Brendan CORMACK
Viale Regina Elena, 299                             Molecular Biology and Genetics
Rome IT 161, ITALY                                  Johns Hopkins University School of Medicine
Tel : +39 (0)6 4990 6135                            725 N. Wolfe St.
Fax : +39 (0)6 4938 7183                            Baltimore MD 21210, USA
Mail: cassone@iss.it                                Tel : 41061444 923
Web : www.iss.it                                    Fax : 4105026 718
                                                    Mail: bcormack@jhmi.edu
Jiangye CHEN
State Key Laboratory of Molecular Biology           Jaime CORREA-BORDES
Institute of Biochemistry and Cell Biology,         Microbiology
SIBS, CAS                                           Universidad de Extremadura
320 Yue-yang Road                                   Avda Elvas s/n
Shanghai 200031, China                              Badajoz 6071, Spain
Tel : 86-21-54921251                                Tel : +34 924289300 ext 6874
Fax : 86-21-54921011                                Fax : 34924289 300
Mail: jychen@sibs.ac.cn                             Mail: jcorrea@unex.es
Web : http://www.sibs.ac.cn/
                                                    Sofia COSTA-DE-OLIVEIRA
Francesco CITIULO                                   Microbiology
Dublin Dental School & Hospital                     Faculty of Medicine
Trinity College Dublin                              Alameda Prof Hernani Monteiro
Lincoln Place                                       Porto 4200, Portugal
Dublin 2, Ireland                                   Tel : 351225513662
Tel : +353 (0)1 612 7275                            Fax : 351225513662
Fax : + 353 (0)1 612 7295                           Mail: sqcoliveira@yahoo.com
Mail: francesco.citiulo@dental.tcd.ie
                                                    Maria C. COSTANZO
Toni CIUDAD                                         Genetics
Departamento de MicrobiologÌa                       Stanford University School of Medicine
Universidad de Extremadura                          Stanford University
Avda. Elvas s/n                                     Stanford CA 94305, USA
Badajoz 6006, Spain                                 Tel : 1-650-498-6012
Tel : 34924289 428                                  Fax : 1-650-724-3701
Fax : 34924289 428                                  Mail: maria@genome.stanford.edu
Mail: aciudad@unex.es                               Web : http://www.candidagenome.org/




                                              211
Alix COSTE                                           Piet DE GROOT
Microbiology Institute                               SILS-Mass Spectrometry of
University Hospital Lausanne                         Biomacromolecules
rue du Bugnon 48                                     University of Amsterdam
Lausanne 1007, Switzerland                           Nieuwe Achtergracht 166
Tel : + 41 (0)21 314 40 61 or 51                     Amsterdam 1018 WV, The Netherlands
Fax : +41 (0)21 314 40 60                            Tel : +31 (0)20 5257053
Mail: alix.coste@chuv.ch                             Fax : +31 (0)20 5257056
                                                     Mail: P.W.J.deGroot@uva.nl
Melanie T. CUSHION
Internal Medicine, Division of Infectious            Christophe D'ENFERT
Diseases                                             Unité Biologie et Pathogénicité Fongiques
University of Cincinnati College of Medicine         Institut Pasteur
231 Albert Sabin Way                                 25 rue du Docteur Roux
Cincinnati OH 45140-0560, USA                        Paris 75015, France
Tel : +1 (513) 861 3100 ext 4417                     Tel : +33 1 40 61 32 57
Fax : +1 (513) 475 6415                              Fax : +33 1 45 68 89 38
Mail: melanie.cushion@uc.edu                         Mail: denfert@pasteur.fr
Web :                                                Web : http://www.pasteur.fr/bpf
http://www.pathobiology.uc.edu/html/disea_im
mun_cushion.html                                     Chen DING
                                                     School of Biomolecular & Biomedical Science
Neelam DABAS                                         Conway Institute, University College Dublin
Institut f‚r Molekulare Infektionsbiologie           Belfield
Universit‰t W¸rzburg                                 Dublin D 4, Turkey
Rˆntgenring 11                                       Tel : +353 1 7166838
W‚rzburg 97070, Germany                              Fax : +353 1 2837211
Tel : 49931312 127                                   Mail: chen.ding@ucd.ie
Fax : 49931312 578
Mail: Neelam.Dabas@mail.uni-wuerzburg.de             Leigh DINI
                                                     Parasitology Reference Unit
Albert DE BOER                                       National Institute for Communicable Diseases
Medical Microbiology                                 1 Modderfontein Road
University of Gˆttingen                              Sandringham 2192, South Africa
Kreuzbergring 57                                     Tel : +27 (0) 11 555 0311
Gˆttingen 37075, Germany                             Fax : +27 (0) 11 555 0446
Tel : +49 (0) 551 39 5848                            Mail: leighd@nicd.ac.za
Fax : +49 (0) 551 39 5861                            Web : www.nicd.ac.za
Mail: aboer@gwdg.de
                                                     Ben DISTEL
Katrijn DE BRUCKER                                   Department of Medical Biochemistry
Department of Molecular Microbiology                 Academic Medical Center
VIB, KULeuven                                        Meibergdreef 15
Kasteelpark Arenberg 31                              Amsterdam 1105 AZ, The Netherlands
Leuven 3000, Belgium                                 Tel : +31-(0)20 5665127
Tel : +32 (0)16 32 1500                              Fax : +31-(0)20 6915519
Fax : +32 (0)16 32 1979                              Mail: b.distel@amc.uva.nl
Mail: katrijn.debrucker@bio.kuleuven.be
Web : http://bio.kuleuven.be/mcb/




                                               212
Marija DUKALSKA                                    Arnaud FIRON
Molecular Biotechnology                            Fungal Biology and Pathogenicity
Fraunhofer Institute                               Institut Pasteur
Nobelstrasse 12                                    25 rue du Docteur Roux
Stuttgart 70569, Germany                           Paris 75015, France
Tel : +49 (0)711 970 4171                          Tel : +33 (0)1 4568 8205
Fax : +49 (0)711 970 4200                          Fax : +33 (0)1 4568 8938
Mail: Marija.Dukalska@igb.fhg.de                   Mail: afiron@pasteur.fr
Web : www.igb.fraunhofer.de
                                                   Michael FLEISCHHACKER
Brice ENJALBERT                                    Med. Klinik m.S. Onkologie/H‰matologie
Aberdeen Fungal Group                              Med. CharitÈ-Universit‰tsmedizin Berlin
Institute of Medical Sciences                      CharitÈplatz 1
University of Aberdeen                             Berlin 10117, Germany
Aberdeen AB25 2ZD, United Kingdom                  Tel : +4930 450 51 33 04
Tel : -557068                                      Fax : +4930 450 51 39 64
Fax : -557024                                      Mail: michael.fleischhacker@charite.de
Mail: brice.enjalbert@abdn.ac.uk
                                                   Chantal FRADIN
Victor FERNANDEZ                                   Inserm U799
Parasitology, Mycology and Environmental           FacultÈ de mÈdecine
Microbiology                                       Place de Verdun
Swedish Institute for Infectious Disease           Lille 59045, France
Control                                            Tel : + 33 (0) 3 20 62 34 21
Nobelsvaeg 18                                      Fax : + 33 (0)3 20 62 34 16
Solna 17182, Sweden                                Mail: cfradin@univ-lille2.fr
Tel : +46 (0)8 457 2553
Fax : +46 (0)8 31 84 50                            Ingrid FROHNER
Mail: victor.fernandez@smi.ki.se                   Medical Biochemistry
Web : http://www.smittskyddsinstitutet.se/         Medical University, Max F. Perutz
                                                   Laboratories
Sélène FERRARI                                     Dr. Bohrgasse 9/2
Institute of Microbiology                          Vienna A 1020, Austria
University Hospital (CHUV)                         Tel : 0043 1 4277 61818
Rue du Bugnon 48                                   Fax : 0043 1 4277 9618
Lausanne 1011, Switzerland                         Mail: ingrid.frohner@meduniwien.ac.at
Tel : +41 21 314 40 61
Fax : +41 21 314 40 60                             Toni GABALDON
Mail: selene.ferrari@chuv.ch                       Bioinformatics
                                                   CIPF
Alessandro FIORI                                   Autopista del Saler, 16
Faculty of Life Sciences                           Valencia 46013, Spain
University of Manchester                           Tel : +34 96 328 9680 (ext 1006)
Oxford Road                                        Fax : +34 96 328 9701
Manchester M13 9PT, United Kingdom                 Mail: tgabaldon@cipf.es
Tel : +44 (0)161 275 1580                          Web : http://bioinfo.cipf.es/tgabaldon
Fax : +44 (0)161 275 1505
Mail: Alessandro.Fiori@manchester.ac.uk            Attila GACSER
                                                   Department of Medicine, Infectious Diseases
                                                   Albert Einsten College of Medicine (AECOM)
                                                   1300 Morris Park Ave
                                                   New York NY 10461, USA
                                                   Tel : 17184302 993
                                                   Fax : 17184308 968
                                                   Mail: gacsera@gmail.com


                                             213
                                                     Steven GILES
Ana GARCERA                                          Biomolecular Chemistry
Department of Basic Medical Sciences,                University of Wisconsin-Madison
Faculty of Medicine                                  687 Medical Sciences Center, 1300 University
University of Lleida                                 Ave.
Montserrat Roig 2                                    Madison WI 53562, USA
Lleida 25008, Spain                                  Tel : (608) 265.5689
Tel : 34973702409                                    Fax : (608) 262.5253
Fax : 34973702426                                    Mail: ssgiles@wisc.edu
Mail: ana.garcera@cmb.udl.es                         Web :
Web : http://www.udl.es/                             http://www.bmolchem.wisc.edu/faculty/hull.ht
                                                     ml
Alexander GEHRKE
Molecular&Applied Microbiology                       Gustavo GOLDMAN
Leibniz Institute for Nat.Product                    Ciencias Farmaceuticas
Research&Inf. Biology                                FCFRP, Universidade de Sao Paulo
Beutenbergstr 11A                                    Av. do Cafe S/N
Jena 7745, Germany                                   Ribeirao Preto SP 14040903, BRAZIL
Tel : 493641656825                                   Tel : +55(16)36024280
Fax : 493641656835                                   Fax : +55(16)36024280
Mail: alexander.gehrke@hki-jena.de                   Mail: ggoldman@usp.br
Web : www.hki-jena.de                                Web : goldman.fcfrp.usp.br

Guri GIAEVER                                         Teresa GONCALVES
Pharmaceutical Sciences and Medical Genetics         Centre for Neurosciences and Cell Biology
University of Toronto                                Institute of Microbiology, Faculty of Medicine
CCBR 160 College St.                                 Universidade de Coimbra, Rua Larga
Toronto On M5S3E1, Canada                            Coimbra 3004-504, Portugal
Tel : 4169787182                                     Tel : -239834378
Fax : 4169788287                                     Fax : -239826447
Mail: guri.giaever@utoronto.ca                       Mail: tmfog@ci.uc.pt
Web : http://chemogenomics.med.utoronto.ca/
                                                     Neil GOW
Concha GIL                                           School of Medical Sciences
Microbiology                                         University of Aberdeen
Complutense University                               Institute of Medical Sciences, Foresterhill
Plaza de Ramón y Cajal s/n                           Aberdeen AB25 2ZD, United Kingdom
Madrid 28040, Madrid                                 Tel : -557059
Tel : 34-91-3941744                                  Fax : -557024
Fax : 34-91-3941745                                  Mail: n.gow@abdn.ac.uk
Mail: conchagil@farm.ucm.es
Web : http://www.ucm.es/info/mfar/                   Lakshmi GOYAL
                                                     Editorial
Tsvia GILDOR                                         Cell Press
Molecular Microbiology                               600 Technology Square
Technion, Israel                                     Cambridge MA 2139, USA
Efron 2                                              Tel : 8578911 909
Haifa IL 31096, Israel                               Fax : 6173972 810
Tel : (0)-972-4-8295258                              Mail: lgoyal@cell.com
Fax : 972-4-8295254
Mail: tsvia@tx.technion.ac.il




                                               214
Sophie GOYARD                                       Ken HAYNES
Fungal Biology and Pathogenicity                    Molecular Microbiology & Infection
Institut Pasteur                                    Imperial College London
25 rue du Docteur Roux                              Exhibition Road
Paris 75015, France                                 London SW7 2AZ, UK
Tel : +33 (0)1 4568 8817                            Tel : +44 (0) 20 7594 2072
Fax : 33 (0)1 4568 8938                             Fax : +44 (0) 20 8383 3394
Mail: goyard@pasteur.fr                             Mail: k.haynes@imperial.ac.uk

Christa GREGORI                                     Joseph HEITMAN
Medical Biochemistry                                Molecular Genetics and Microbiology
Medical University of Vienna, Max F. Perutz         Duke University
Laboratories                                        Research Drive
Dr. Bohrgasse 9/2                                   Durham NC 27710, USA
Vienna 1030, Austria                                Tel : 9196842 824
Tel : +43 (0)1 4277 61818                           Fax : 9196855 458
Fax : +43 (0)1 4277 9618                            Mail: heitm001@duke.edu
Mail: christa.gregori@meduniwien.ac.at              Web :
                                                    http://mgm.duke.edu/microbial/mycology/heit
Per HAGBLOM                                         man/
Parasitology, Mycology and Environmental
Microbiology                                        Mariana HENRIQUES
Swedish Institute for Infectious Disease            IBB-CEB
Control                                             University of Minho
Nobels v‰g 18                                       Campus Gualtar
Solna 17182, Sweden                                 Braga 4710-057, Portugal
Tel : +46 (0)8 4572548                              Tel : 351253604418
Fax : +46 (0)8 318450                               Fax : 351253678996
Mail: per.hagblom@smi.ki.se                         Mail: mcrh@deb.uminho.pt
Web : www.smittskyddsinstitutet.se
                                                    Rosa HERNANDEZ
Thomas HARTMANN                                     MBT
Institut fuer Mikrobiologie und Genetik             Fraunhofer IGB
Georg August Universitaet Goettingen                Nobelstrasse, 12
Grisebachstrasse 8                                  Stuttgart BW 70569, Germany
Goettingen 37077, Germany                           Tel : +49(0)7119704048
Tel : 0049 551 39 3821                              Fax : +49(0)7119704200
Fax : 0049-551-393330                               Mail: rhe@igb.fraunhofer.de
Mail: hartmann.tom@gmail.com                        Web : http://www.igb.fraunhofer.de/

Mike HASENBERG                                      Ekkehard HILLER
Nachwuchsforschergruppe Immundynamik                Fraunhofer IGB
Helmholtz Zentrum f‚r Infektionsforschung           Nobelstrasse 12
Inhoffenstrafle 7                                    Stuttgart 70569, Germany
Braunschweig 38124, Germany                         Tel : +49 (0)711 970 4050
Tel : +49 (0) 531 6181 3132                         Fax : +49 (0)711 970 4200
Fax : +49 (0) 531 6181 3199                         Mail: hiller@igb.fraunhofer.de
Mail: mike.hasenberg@gbf.de                         Web : http://www.igb.fhg.de
Web : http://www.helmholtz-
hzi.de/de/forschergruppen/zell_und_immunbio
logie/immundynamik/




                                              215
Denes HNISZ                                         Bernhard HUBE
Medical Biochemistry                                Micobial Pathogenicity Mechanisms
Max F. Perutz Laboratories                          Hans Knoell Institute
Dr Bohr-gasse 9/2                                   Beutenbergstr. 11A
Vienna 1030, Austria                                Jena 7745, Germany
Tel : 431427761812                                  Tel : +49 (0)3641 65-6881
Fax : 43142779618                                   Fax : +49 (0)3641 65-6882
Mail: denes.hnisz@meduniwien.ac.at                  Mail: bernhard.hube@hki-jena.de
                                                    Web : www.hki-jena.de
Ann HOLMES
Oral Sciences                                       Christina HULL
University of Otago                                 Dept. of Biomolecular Chemistry
310 Great King St                                   University of Wisconsin, Madison
Dunedin 9054, New Zealand                           1300 University Ave. 587 MSC
Tel : +64 (0)3 479 7435                             Madison WI 53706, United States
Fax : +64 (0)3 479 7078                             Tel : 608-265-5441
Mail: ann.holmes@otago.ac.nz                        Fax : 608-262-5253
                                                    Mail: cmhull@wisc.edu
Samantha HOOT
Pathobiology                                        Dervla ISAAC
University of Washington/Seattle Biomedical         Microbiology and Immunology
307 Westlake Ave Suite 500                          University of California - San Francisco
Seattle WA 98109, USA                               (UCSF)
Tel : 4257501560                                    513 Parnassus Ave, Room S-472
Fax : 2062567229                                    San Francisco CA 94143, USA
Mail: sam.hoot@sbri.org                             Tel : 011-1-415-502-4810
                                                    Fax : 011-1-415-476-8201
Hannah HOPE                                         Mail: Dervla.Isaac@ucsf.edu
Institute of Signalisation Developmental
Biology and Cancer                                  Mette D. JACOBSEN
Parc Valrose                                        Aberdeen Fungal Group
Nice 6108, France                                   University of Aberdeen
Tel : 0033 (0)4 92 07 6465                          Foresterhill
Fax : 0033 (0)4 92 07 6466                          Aberdeen AB25 2ZD, United Kingdom
Mail: hope@unice.fr                                 Tel : 441224555 888
Web : http://www.unice.fr/isdbc/                    Fax : 441224555 844
                                                    Mail: m.d.jacobsen@abdn.ac.uk
Olga HRUSKOVA-HEIDINGSFELDOVA
Gilead Sciences Research Center                     Guilhem JANBON
Institute of Organic Chemistry and                  Mycologie MolÈculaire
Biochemistry                                        Institut Pasteur
Flemingovo n. 2                                     25 rue du Dr Roux
Prague 166 10, Czech Republic                       Paris 75015, France
Tel : 420220183 249                                 Tel : 33 (0)145688356
Fax : 420220183 578                                 Fax : 33 (0)145688420
Mail: olga-hh@uochb.cas.cz                          Mail: janbon@pasteur.fr
Web : www.uochb.cas.cz




                                              216
JOHNSON Alexander                                     Anna KOLECKA
Microbiology and Immunology                           Department of Microbiology and Virology
UCSF                                                  Faculty of Natural Sciences, Comenius
600 16th Street                                       University
San Francisco CA 94158 (USA)
                                                      Mlynska dolina B2
Phone: 415 476 8783
FAX: 415 502 4315
                                                      Bratislava SR 84215, Slovakia
e-mail: ajohnson@cgl.ucsf.edu                         Tel : +421 2 602 96 636
                                                      Fax : +421 2 654 29 064
Laura Ann JONES                                       Mail: kolecka@fns.uniba.sk
Molecular biology and Biotechnology                   Web : -
University of Sheffield
Western Bank                                          James KONOPKA
Sheffield S10 2TN, United Kingdom                     Dept. Molecular Genetics and Microbiology
Tel : +44 (0)114 2222748                              State University of New York
Fax : +44 (0)114 2222748                              Room 130 Life Sciences Bldg.
Mail: mbp05laj@sheffield.ac.uk                        Stony Brook NY 11794-5222, USA
Web : www.shef.ac.uk                                  Tel : +1 631 632-8715
                                                      Fax : +1 631 632-9797
Dariusz KAWECKI                                       Mail: jkonopka@ms.cc.sunysb.edu
General Surgery and Transplantation / Medical
Microbiology                                          Daniel KORNITZER
Medical University                                    Molecular Microbiology
Chalubinski 5                                         Technion Faculty of Medecine
Warsaw 02-004, Poland                                 2, Efron St.
Tel : +48 022 622-00-28                               Haifa 31096, Israel
Fax : +48 022 628-27-39                               Tel : +972 (0)4 8295258
Mail: dkawecki@o2.pl                                  Fax : +972 (0)4 8295254
Web : www.am.edu.pl                                   Mail: danielk@tx.technion.ac.il

Nancy KELLER                                          Lucie KRAIDLOVA
Plant Pathology and Medical Microbiology              Department of Membrane Transport
Immunology                                            Institut of Physiology
University of Wisconsin                               Videnska 1083
1630 Linden Drive                                     Prague 14220, Czech Republic
Madison wi 53706, United States                       Tel : 420241061 111
Tel : 608 262-9795                                    Fax : 420241062 488
Fax : (608) 263-2626                                  Mail: kraidlova@biomed.cas.cz
Mail: npk@plantpath.wisc.edu
Web :                                                 Ephie KRANEVELD
http://www.plantpath.wisc.edu/fac/npk.htm             Cariology Endontology Pedodontology
                                                      Academic Centre for Dentistry Amsterdam
bruce KLEIN                                           Louwesweg 1
pediatrics and medical microbiology                   Amsterdam 1066 EA, The Netherlands
university of wisconsin-madison                       Tel : +31 20 5188596
600 highland avenue                                   Fax : +31 20 6692881
madison wi 53792, usa                                 Mail: E.Kraneveld@acta.nl
Tel : 608-263-9217                                    Web : www.acta.nl
Fax : 608-263-6210
Mail: bsklein@wisc.edu
Web :
http://www.medmicro.wisc.edu/department/fac
ulty/klein.html




                                                217
Yannick KRAUKE                                        German LARRIBA
Dept. Membrane Transport                              MicrobiologÌa
Institute of Physiology AS CR, v.v.i.                 Universidad de Extremadura
Videnska 1083                                         Avda de Elvas s/n
Prague 142 20, Czech Republic                         Badajoz 6071, Spain
Tel : 420241062120                                    Tel : 34924289 428
Fax : 420296442498                                    Fax : 34924289 428
Mail: krauke@biomed.cas.cz                            Mail: glarriba@unex.es
                                                      Web : http://www.unex.es
Sona KUCHARIKOVA
Microbiology and Virology                             Ulrich LERMANN
Faculty of Natural Sciences, Comenius                 Institut f‚r Molekulare Infektionsbiologie
University                                            Universit‰t W¸rzburg
Mlynska dolina B-2                                    Rˆntgenring 11
Bratislava SR 84215, Slovakia                         W‚rzburg 97070, Germany
Tel : 00421 2 60296636                                Tel : +49 (0) 931 312127
Fax : 00421 2 65429064                                Fax : +49 (0) 931 312578
Mail: sonakucharikova@yahoo.com                       Mail: u.lermann@mail.uni-wuerzburg.de
Web : -
                                                      Franziska LESSING
Karl KUCHLER                                          molecular and applied microbiology
Max F. Perutz Laboratories                            Leibniz Inst. for natural prod. research +
Medical University Vienna                             infection biology
Dr. Bohr-Gasse 9/2                                    Beutenberg str. 11a
Vienna A-1030, Austria                                Jena 7745, Germany
Tel : -66042                                          Tel : +49 (0) 3641 656817
Fax : -13853                                          Fax : +49 (0) 3641 656603
Mail: karl.kuchler@meduniwien.ac.at                   Mail: franziska.lessing@hki-jena.de
Web :
http://www.meduniwien.ac.at/medbch/MolGen             Stuart LEVITZ
/kuchler/                                             Infectious Diseases
                                                      University of Massachusetts
Leslie LAFORET                                        364 Plantation Street
Department of Microbiology and Ecology                Worcester MA 1605, USA
University of Valencia                                Tel : 508-856-1525
Vicente andres estelles av. n/n                       Fax : 5088561828
Burjassot-Valencia 46100, Spain                       Mail: stuart.levitz@umassmed.edu
Tel : 34 96 386 46 57                                 Web :
Fax : 34 96 386 46 57                                 http://www.umassmed.edu/ivp/faculty/levitz.cf
Mail: Leslie.Laforet@uv.es                            m
Web :
http://centros.uv.es/web/departamentos/D275/i         Elena LINDEMANN
ngles/                                                MBT
                                                      Fraunhofer IGB
Rachel LANE                                           Nobelstr. 12
Molecular Biology and Biotechnology                   Stuttgart 70569, Germany
University of Sheffield                               Tel : 497119704 145
Firth Court, Western Bank                             Fax : 497119704 200
Sheffield S10 2TN, United Kingdom                     Mail: elena.lindemann@igb.fraunhofer.de
Tel : +44(0)II42222748
Fax : +44(0)1142222748
Mail: MBP04RFL@SHEFFIELD.AC.UK




                                                218
Teresa LIU                                            Raquel MARTINEZ-LOPEZ
Clinical Pharmacy                                     MicrobiologÌa II
University of Tennessee Health Science Center         Facultad de Farmacia, UCM
50 N. Dunlap St. WPT Rm 304                           Plaza de RamÛn y Cajal S/N
Memphis TN 38103, USA                                 Madrid 28040, Spain
Tel : 901-287-5388                                    Tel : +34 91 394 17 55
Fax : 901-287-5036                                    Fax : +34 91 394 17 45
Mail: tliu@utmem.edu                                  Mail: raquelml@farm.ucm.es
                                                      Web : http://www.ucm.es
Per LJUNGDAHL
Department of Cell Biology                            Margarida MARTINS
Wenner-Gren Institute                                 IBB-Institute for Biotechnology and
Stockholm University                                  Bioengineering
Stockholm SE 10691, Sweden                            Universidade do Minho
Tel : 0046 8 164101                                   Campus de Gualtar
Fax : 0046 8 159837                                   Braga 4710-057, Portugal
Mail: plju@wgi.su.se                                  Tel : 351253604 400
Web : http://www.wgi.su.se                            Fax : 351253678 986
                                                      Mail: margarida.martins@deb.uminho.pt
Michael LORENZ
Microbiology and Molecular Genetics                   Emilia MELLADO
The University of Texas Health Science Center         Servicio de Micologia
6431 Fannin                                           Instituto Salud Carlos III
Houston TX 77030, USA                                 Carretera Majadahonda-Pozuelo km2
Tel : (713) 500-7422                                  Majadahonda, Madrid 28220, Spain
Fax : (713) 500-5499                                  Tel : 34918223661
Mail: Michael.Lorenz@uth.tmc.edu                      Fax : 34915097034
Web : http://www.lorenzlab.org                        Mail: emellado@isciii.es
                                                      Web : http://www.isciii.es
Omar LOSS
Molecular Microbiology and Infection                  Céline MILLE
Imperial College London                               Inserm U799
Armstrong Road                                        FacultÈ de mÈdecine
London SW7 2AZ, United Kingdom                        place de verdun
Tel : 00 44 207 534 5293                              Lille 59000, France
Fax : 00 44 207 594 3076                              Tel : +33 (0) 3 20 62 34 15
Mail: o.loss04@imperial.ac.uk                         Fax : + 33 (0)3 20 62 34 16
                                                      Mail: celine.mille@univ-lille2.fr
Olivia MAJER
Medical Biochemistry                                  Isabel MIRANDA
Max F. Perutz laboratories                            Biology and CESAM
Dr. Bohrgasse 9/2                                     University of Aveiro
Vienna 1030, Austria                                  Campus de Santiago
Tel : +43 1 4277 61812                                Aveiro 3810-193, Portugal
Fax : +43 1 4277 9618                                 Tel : 351234370 970
Mail: olivia.majer@meduniwien.ac.at                   Fax : 351234426 408
                                                      Mail: imiranda@bio.ua.pt




                                                219
Estelle MOGENSEN
Mycologie MolÈculaire                               Ntombizamatshali MTSHALI
Institut Pasteur                                    Biology
25 rue du Docteur Roux                              University of Kwa-Zulu Natal
Paris 75015, France                                 22 Carbis Road
Tel : +33 (0)1 40 61 83 56                          Pietermaritzburg 2301, South Africa
Fax : +33 (0)1 45 68 84 20                          Tel : 44(0) 115 9709398
Mail: estelle.mogensen@pasteur.fr                   Fax : 44 (0) 115 3251
Web : http://www.pasteur.fr/                        Mail: 204507919@ukzn.ac.za
                                                    Web : www.ukzn.ac.za
Gary MORAN
Division of Oral Biosciences                        Fritz A. MUHLSCHLEGEL
Dublin Dental School and Hospital                   Department of Biosciences
Lincoln Place                                       University of Kent
Dublin 2, Ireland                                   Giles Lane
Tel : +353 1 6127245                                Canterbury CT27NJ, United Kingdom
Fax : +353 1 6127295                                Tel : 441227823 988
Mail: gpmoran@dental.tcd.ie                         Fax : 441227763 912
                                                    Mail: f.a.muhlschlegel@kent.ac.uk
Zuzana MORANOVA
Department of Microbiology                          Siobhan MULHERN
Palacky University                                  School of Biomolecular and Biomedical
Hnevotinska 3                                       Research
Olomouc 77515, Czech Republic                       Conway Institute
Tel : 420585639 505                                 University College Dublin, Belfield
Fax : 420585632 417                                 Dublin D4, Ireland
Mail: zuzana.moranova@post.sk                       Tel : 00353-(0)1-7166838
                                                    Fax : 00353 (0)1-2837211
Inmaculada MORENO                                   Mail: siobhan.mulhern@ucd.ie
Microbiology and Ecology
Faculty of Pharmacy. University of Valencia         Carol MUNRO
Avda. Vicente Andres Estelles s/n                   School of Medical Sciences
Burjassot- Valencia 46100, Spain                    University of Aberdeen
Tel : 34963544 684                                  Foresterhill
Fax : 34963544 543                                  Aberdeen AB25 2ZD, United Kingdom
Mail: inmaculada.moreno@uv.es                       Tel : +44 (0) 1224555927
                                                    Fax : +44 (0) 1224555844
Joachim MORSCHAUSER                                 Mail: c.a.munro@abdn.ac.uk
Institut f‚r Molekulare Infektionsbiologie          Web :
Universit‰t W¸rzburg                                http://www.abdn.ac.uk/ims/staff/details.php?id
Rˆntgenring 11                                      =c.a.munro
W‚rzburg 97070, Germany
Tel : -313034                                       Julian NAGLIK
Fax : -313460                                       Oral Immunology
Mail: joachim.morschhaeuser@mail.uni-               King's College London
wuerzburg.de                                        St Thomas Street
                                                    London SE1 9RT, United Kingdom
David MOYES                                         Tel : +44 20 7188 4377
Oral Immunology                                     Fax : +44 20 7188 4375
King's College                                      Mail: julian.naglik@kcl.ac.uk
St. Thomas Street
London SE1 9RT, United Kingdom
Tel : 2071884 377
Fax : 2071884 375
Mail: david.moyes@kcl.ac.uk


                                              220
Priyanka NARANG
Junior Research Group Immunodynamics              Suzanne NOBLE
Helmholtz Zentrum f‚r Infektionsforschung         Microbiology and Immunology Department
Inhoffenstrasse 7                                 UCSF
Braunschweig 38124, Germany                       600 16th St., Box 2200
Tel : +49 (0) 531 6181 3132                       San Francisco CA 94143-2200, United States
Fax : +49 (0) 531 6181 3199                       Tel : (001) 415-502-0859
Mail: pna04@helmholtz-hzi.de                      Fax : (001) 415-502-4315
Web : http://www.helmholtz-hzi.de                 Mail: Suzanne.Noble@ucsf.edu

Claire NAULLEAU                                   Leanne O'CONNOR
Fungal Biology and Pathogenicity                  Oral Microbiology
Institut Pasteur                                  Dublin Dental School and Hospital, Trinity
25 rue du Docteur Roux                            College
Paris 75015, France                               Lincoln Place
Tel : +33 (0)1 4061 3126                          Dublin D2, Ireland
Fax : +33 (0)1 4568 8938                          Tel : +353 (0)1 612 7350
Mail: naulleau@pasteur.fr                         Fax : +353 (0)1 612 7295
                                                  Mail: oconnorl@dental.tcd.ie
Susan NICHOLLS
Aberdeen Fungal Group                             Rosario OLIVEIRA
Institute of Medical Sciences                     Department of Biological Engineering
Foresterhill                                      University of Minho
Aberdeen AB25 2ZD, United Kingdom                 Campus Gualtar
Tel : 1224555 888                                 Braga 4710-057, Portugal
Fax : 1224555 844                                 Tel : 351253604419
Mail: s.nicholls@abdn.ac.uk                       Fax : 351253678996
                                                  Mail: roliveira@deb.uminho.pt
William C. NIERMAN
The Institute for Genomic Research                Jason OLIVER
9712 Medical Center Drive                         Targets
Rockville MD 20850, USA                           F2G Ltd
Tel : 301-795-7559                                PO Box 1, Lankro Way, Eccles
Fax : 301-838-3009                                Manchester M30 0BH, United Kingdom
Mail: wnierman@tigr.org                           Tel : +44 (0) 161 275 1270
Web : www.tigr.org                                Fax : +44 (0) 161 785 1273
                                                  Mail: jasonoliver@f2g.com
Masakazu NIIMI                                    Web : http://www.f2g.com/
Deaprtment of Bioactive Molecules
National Institute of Infectious Diseases         Ritu PASRIJA
1-23-1 Toyama, Shinjuku-ku                        School of Life Sciences
Tokyo 162-8640, JAPAN                             Jawaharlal Nehru University
Tel : +81 (0)3 5285 1111                          New Delhi
Fax : +81 (0)3 5285 1272                          New Delhi 110067, India
Mail: niimi@nih.go.jp                             Tel : -7099
                                                  Fax : -9672
Elissavet NIKOLAOU                                Mail: ritupasrija@yahoo.com
Molecular and Cell Biology
Institute of Medical Sciences
Foresterhill
Aberdeen AB25 2ZD, United Kingdom
Tel : -557068
Fax : -557024
Mail: e.nikolaou@abdn.ac.uk
Web : www.abdn.ac.uk


                                            221
Tatiana PEREIRA                                     Jennifer REEDY
Cariology Endontology Pedodontology                 Molecular Genetics and Microbiology
Academic Centre for Dentistry Amsterdam             Duke University
Louwesweg 1                                         320 CARL Bldg, Research Drive, Box 3546
Amsterdam 1066 EA, The Netherlands                  Durham NC 27710, USA
Tel : +31 20 5188596                                Tel : 19196843 036
Fax : +31 20 6692881                                Fax : 19196845 458
Mail: T.Pereira@acta.nl                             Mail: reedy004@mc.duke.edu
Web : www.acta.nl
                                                    Oliver REUSS
Emmanuelle PINJON                                   Institut f‚r Molekulare Infektionsbiologie
Department of Oral Biosciences                      Universit‰t W¸rzburg
Dublin Dental Hospital                              Rˆntgenring 11
Lincoln Place                                       W‚rzburg 97070, Germany
Dublin 2, Ireland                                   Tel : +49 (0) 931 312127
Tel : +353 (0)1 612 7350                            Fax : +49 (0) 931 312578
Fax : +353 (0)1 612 7295                            Mail: oliver.reuss@mail.uni-wuerzburg.de
Mail: emmanuelle.pinjon@dental.tcd.ie
                                                    Elisabete RICARDO
Aida PITARCH                                        Microbiology
Microbiology II                                     Faculty of Medicine Porto University
Faculty of Pharmacy, Complutense University         Alameda Prof. Hernani Monteiro
of Madrid                                           Porto 4200-319, Portugal
Plaza RamÛn y Cajal, s/n                            Tel : 351225513 662
Madrid 28040, Spain                                 Fax : 351225513 662
Tel : +34 91 394 1755                               Mail: betaricardo@yahoo.com
Fax : +34 91 394 1745
Mail: apitavel@farm.ucm.es                          Marketa RICICOVA
Web : http://www.ucm.es/                            Department of Molecular Microbiology
                                                    VIB, Katholieke Universiteit Leuven
Daniel POULAIN                                      Kasteelpark Arenberg 31
Inserm unit 799 Physiopathologie des                Leuven-Heverlee 3001, Belgium
Candidoses                                          Tel : +32 (0)16 32 1500
Faculty of Medicine/University Hospital             Fax : +32 (0)16 32 1979
Place Verdun                                        Mail: Marketa.Ricicova@bio.kuleuven.be
Lille 59, FRANCE                                    Web : http://bio.kuleuven.be/mcb
Tel : +33 (0)3 2062 3420
Fax : +33 (0)3 2062 3416                            Alexandra RODAKI
Mail: dpoulain@univ-lille2.fr                       Aberdeen Fungal Group
                                                    University of Aberdeen
Rajendra PRASAD                                     Forresterhill
School of Life Sciences                             Aberdeen AB25 2ZD, United Kingdom
Jawaharlal Nehru University                         Tel : 441224555898
School of Life Sciences                             Fax : 4,41222E+12
New Delhi 110067, India                             Mail: a.rodaki@abdn.ac.uk
Tel : -26704429                                     Web : www.abdn.ac.uk
Fax : -26717001
Mail: rp47@hotmail.com                              Marc ROEHM
                                                    MBT
                                                    Fraunhofer IGB
                                                    Nobelstr. 12
                                                    Stuttgart 70569, Germany
                                                    Tel : 497119704 171
                                                    Fax : 497119704 200
                                                    Mail: mro@igb.fraunhofer.de


                                              222
                                           Silvia SANDINI
Andreas ROETZER                            Infectious, Parasitic and Immuno-mediated
Dpt. of Biochemistry                       Diseases
MFPL - University of Vienna                Istituto Superiore di Sanit‡
Dr. Bohrgasse 9/5                          viale Regina Elena 299
Vienna 1030, Austria                       Rome 161, Italy
Tel : +43 (0)1 4277 52805                  Tel : +39 (0)6 4990 2369
Fax : +43 (0)1 4277 9528                   Fax : +39 (0)6 4938 7112
Mail: andreas.roetzer@univie.ac.at         Mail: sandini@iss.it
Web : http://www.mfpl.ac.at/               Web : http://www.iss.it

Tristan ROSSIGNOL                          Dominique SANGLARD
Fungal Biology and Pathogenicity           Institute of Microbiology
Institut Pasteur                           University Hospital Lausanne
25 rue du Docteur Roux                     Rue du Bugnon 48
Paris 75015, France                        Lausanne 1011, Switzerland
Tel : +33 (0) 1 45 68 82 05                Tel : +41 21 3144083
Fax : +33 (0)1 4568 8938                   Fax : +41 21 3144060
Mail: tristan.rossignol@pasteur.fr         Mail: Dominique.Sanglard@chuv.ch
Web : http://www.pasteur.fr/bpf            Web : http://www.chuv.ch/imul/

Markus RUHNKE                              Marlene SANTOS
Medicine                                   Department of Biology
CharitÈ university medicine                University of Minho
CharitÈplatz 1                             Campus de Gualtar
Berlin 10117, Germany                      Braga 4710-057, Portugal
Tel : 4,93045E+12                          Tel : 351253604310
Fax : 4,93045E+12                          Fax : 351253678980
Mail: markus.ruhnke@charite.de             Mail: mmarlenes@gmail.com
Web : www.charite.de
                                           Annika SCHEYNIUS
Steffen RUPP                               Clinical Allergy Research Unit, Dept of
Molecular Biotechnology                    Medicine Solna
Fraunhofer IGB                             Karolinska Institutet
Nobelstr. 12                               Karolinska University Hospital Solna L2:04
Stuttgart 70569, Germany                   Stockholm 171 76, Sweden
Tel : +49 (0) 711 970 4045                 Tel : +46 8 5177 5934
Fax : +49 (0) 711 970 4200                 Fax : +46 8 335724
Mail: rupp@igb.fhg.de                      Mail: annika.scheynius@ki.se
Web : www.fraunhofer.igb.de
                                           Petra SCHLICK
Chelsea SAMANIEGO                          Antigen Discovery
Pathobiology                               Intercell AG
University of Washington/SBRI              6 Campus Vienna Biocenter
307 Westlake ave N                         Vienna 1030, Austria
Seattle WA 98109, USA                      Tel : +43 (0)1 20620 212
Tel : 206-256-7156                         Fax : +43 (0)1 20620 805
Fax : 206-256-7229                         Mail: pschlick@intercell.com
Mail: chelsea.samaniego@sbri.org




                                     223
André SCHMIDT                                        Christine SELANDER
Molecular and Applied Microbiology                   Department of Medicine, Clinical Allergy
Hans-Knoell-Institute / Friedrich-Schiller-          Research Unit
University Jena                                      Karolinska Institutet
Beutenbergstr. 11a                                   Karolinska University Hospital Solna
Jena 7745, Germany                                   Stockholm 171 76, Sweden
Tel : 493641656 858                                  Tel : +46 (0)8 517 76697
Fax : 493641656 603                                  Fax : +46 (0)8 33 57 24
Mail: andre.schmidt@hki-jena.de                      Mail: Christine.Selander@ki.se
Web : www.hki-jena.de
                                                     Elena SHEKHOVTSOVA
Inga SCHMIDT                                         Immunology
Institut f‚r Mikrobiologie                           Shemyakin & Ovchinnikov Institute of
Heinrich-Heine-Universit‰t                           Bioorganic chemistry
Universit‰tsstrasse 1                                Mikluho-Maklaya, 16/10
D‚sseldorf 40225, Germany                            Moscow 117997, Russian Federation
Tel : +49 (0)211 8114833                             Tel : +7 (495) 330-40-11
Fax : +49 (0)211 8115370                             Fax : +7 (495) 330-40-11
Mail: Inga.schmidt@uni-duesseldorf.de                Mail: shehovcova_elena@mail.ru

Bettina SCHULZ                                       Anita SIL
Molekulare Infektionsdiagnostik                      Microbiology and Immunology
CharitÈ Universit‰tsmedizin Berlin                   University of California San Francisco
CharitÈplatz 1                                       513 Parnassus, S-469, UC Medical Center
Berlin 10117, Germany                                San Francisco CA 94143-0414, United States
Tel : +49 (0) 30 450 513376                          Tel : 415 502-1805
Fax : +49 (0) 30 450 513976                          Fax : 415 476-8201
Mail: b.schulz@charite.de                            Mail: sil@cgl.ucsf.edu
Web : www.charite.de                                 Web : http://www.ucsf.edu/micro/sil/index.htm

Mark SCHUTTE                                         Sonia SILVA
Department of biotechnology and biochemistry         Biological Engineering
TU Braunschweig                                      University of Minho
Spielmannstrasse 7                                   Campus de Gualtar
Braunschweig 38106, Germany                          Braga 4710-057, Portugal
Tel : +49 (0) 53 13 91 57 60                         Tel : 351253604 409
Fax : +49.531.391.5763                               Fax : 351253678 986
Mail: ma.schuette@tu-bs.de                           Mail: soniasilva@deb.uminho.pt
Web : www.tu-braunschweig.de/bbt
                                                     Franck Alexandre SKRZYPEK
Tobias SCHWARZMULLER                                 Experimental Medicine and Biochemical
Medical Biochemistry                                 Sciences
Medical University of Vienna, MFPL                   University of Perugia
Dr-Bohr Gasse 9/2                                    Via Del Giochetto
Vienna 1030, Austria                                 Perugia 6126, Italy
Tel : +43(0)1 4277 61818                             Tel : +39 (0)75 / 5857407
Fax : +43(0)1 4277 9618                              Fax : +39 (0)75 / 5857407
Mail:                                                Mail: frafabs@hotmail.com
Tobias.Schwarzmueller@meduniwien.ac.at




                                               224
Jacky SNOEP                                       Bram STYNEN
Department of Biochemistry                        VIB - Laboratory of Molecular Cell Biology
University of Stellenbosch                        Catholic University of Leuven
Private Bag X1                                    Kasteelpark Arenberg 31
Stellenbosch 7600, South Africa                   Leuven-Heverlee 3001, Belgium
Tel : 27218085862                                 Tel : +32 (0)16 32 19 50
Fax : 27218085863                                 Fax : +32 (0)16 32 19 79
Mail: jls@sun.ac.za                               Mail: bram.stynen@bio.kuleuven.be
                                                  Web : http://bio.kuleuven.be/mcb
Kai SOHN
MBT                                               Peter SUDBERY
Fraunhofer IGB                                    Molecular Biology and Biotechnology
Nobelstr. 12                                      Sheffield University
Stuttgart 70569, Germany                          Western Bank
Tel : 497119704 055                               Sheffield S10 2TN, United Kingdom
Fax : 497119704 200                               Tel : +44 (0)114 2226186
Mail: kai.sohn@igb.fraunhofer.de                  Fax : +44 (0)114 2222800
                                                  Mail: P.Sudbery@shef.ac.uk
Martin J SPIERING
Microbiology Research Unit, Dublin Dental         Essa SULEMAN
School & Hospital                                 Department of Biochemistry and
Trinity College Dublin                            Microbiology
Lincoln Place                                     Nelson Mandela Metropolitan University
Dublin 2, Ireland                                 P.O. Box 77000
Tel : +353 (0)1 612 7260                          Summerstrand Port Elizabeth 6000, South
Fax : +353 (0)1 6127295                           Africa
Mail: spierinm@tcd.ie                             Tel : +27 (0)41 504 2608
                                                  Fax : +27 (0)41 504 2814
Peter STAIB                                       Mail: essa.suleman@nmmu.ac.za
Service de Dermatologie
Centre Hospitalier Universitaire Vaudois          John SYNNOTT
Avenue de Beaumont 29                             UCD School of Biomolecular and Biomedical
Lausanne 1011, Switzerland                        Science
Tel : +41 (0) 21 31 46876                         Conway Institute
Fax : +41 (0) 21 31 40378                         University College Dublin, Belfield
Mail: Peter.Staib@chuv.ch                         Dublin Dublin 4, Ireland
                                                  Tel : +353-(0)1-176-6838
Karin STRIJBIS                                    Fax : +353-(0)1-283-7211
Department of Medical Biochemistry                Mail: john.synnott@ucd.ie
Amsterdam Medical Center                          Web : http://www.ucd.ie/conway/cv_72.html
Meibergdreef 15
Amsterdam 1105 AZ, The Netherlands                Driss TALIBI
Tel : 31205665132                                 Eurogentec
Fax : 31306915519                                 Eurogentec Building 2
Mail: K.Strijbis@amc.uva.nl                       Liege Science Park
                                                  Rue Bois Saint-Jean n° 5
                                                  4102 Seraing (BELGIUM)
Joy STURTEVANT
                                                  Phone: +32 4372 7439
Microbiology and Immunology
                                                  FAX: +32 4372 7500
LSUHSC School of Medicine                         e-mail: dr.talibi@eurogentec.com
1901 Perdido St
New Orleans LA 70117, USA
Tel : +1 504-568-6116
Fax : +1 504-568-2918
Mail: jsturt@lsuhsc.edu



                                            225
Koichi TANABE                                        Veronica VESES
Department of Bioactive Molecules                    Molecular and Cell Biology
National Institute of Infectious Diseases            School of Medical Sciences
1-23-1 Toyama, Shinjuku-ku                           Foresterhill
Tokyo 162-8640, Japan                                Aberdeen AB25 2ZD, United Kingdom
Tel : +81 (0)3 5285 1111                             Tel : 441224555888
Fax : +81 (0)3 5285 1272                             Fax : 441224555844
Mail: ktanabe@nih.go.jp                              Mail: v.veses@abdn.ac.uk
                                                     Web : www.abdn.ac.uk
Ana TRAVEN
Molecular Genetics                                   Zuzana VINTEROVA
St. Vincent's Institute                              Gilead Sciences Research Centre
Princes Street                                       Institute of Organic Chemistry and
Fitzroy, Melbourne, Victoria 3065, Australia         Biochemistry AS CR
Tel : +61 (0)3 9288 2480                             Flemingovo nam. 2
Fax : +61 (0)3 9416 2676                             Prague 16610, Czech Republic
Mail: atraven@svi.edu.au                             Tel : +42(0)220183242
Web :                                                Fax : +42(0)220183556
http://www.svi.edu.au/index.cfm?objectID=C           Mail: vinterova@uochb.cas.cz
A28A1CA-A834-C4B3-CF1753DF70F1F896
                                                     Andrea WALTHER
Constantin URBAN                                     Yeast Biology
Cellular Microbiology                                Carlsberg Laboratory
Max Planck Institute for Infection Biology           Gamle Carlsberg Vej 10
CharitÈplatz 1                                       Copenhagen 2500, Denmark
Berlin 10117, Germany                                Tel : 4533275 230
Tel : +49 (0)30 28460 357                            Fax : 4533274 708
Fax : +49 (0)30 28460 301                            Mail: anwa@crc.dk
Mail: curban@mpiib-berlin.mpg.de
Web : http://www.mpiib-berlin.mpg.de/                Huafeng WANG
                                                     State Key Laboratory of Molecular Biology
Patrick VAN DIJCK                                    Institute of Biochemistry and Cell Biology,
Molecular Microbiology, Laboratory of                SIBS, CAS
Molecular Cell Biology                               320 Yue-yang Road
VIB, K.U. Leuven                                     Shanghai 200031, China
Kasteelpark Arenberg 31                              Tel : 86-21-54921152
Leuven 3001, Belgium                                 Fax : 86-21-54921011
Tel : +32(0)16 321512                                Mail: wanghuafeng@sibs.ac.cn
Fax : +32(0)16 321979                                Web : http://www.sibs.ac.cn/
Mail: patrick.vandijck@bio.kuleuven.be
Web : http://bio.kuleuven.be/mcb                     Peter WARN
                                                     Respiratory Medicine
Anna VECCHIARELLI                                    The University of Manchester
Experimental Medicine and Biochemical                1.800 Stopford Building, Oxford Road
Sciences                                             Manchester M13 9PT, United Kingdom
University of Perugia                                Tel : 441616067 215
Via del Giochetto                                    Fax : 441612755 656
Perugia 6126, Italy                                  Mail: peter.warn@manchester.ac.uk
Tel : 390755857 407
Fax : 390755857 407
Mail: vecchiar@unipg.it




                                               226
Kai WEBER
Molekulare Infektionsdiagnostik                      Jae-Hyuk YU
CharitÈ Universit‰tsmedizin Berlin                   Bacteriology
CharitÈplatz 1                                       University of Wisconsin
Berlin 10117, Germany                                1925 Willow Dr.
Tel : +49 (0) 30 450 51 33 04                        Madison WI 53706, USA
Fax : +49 (0) 30 450 51 39 64                        Tel : 1-608-262-4696
Mail: weber_kai@web.de                               Fax : 1-608-263-1114
Web : www.charite.de                                 Mail: jyu1@wisc.edu
                                                     Web : http://www.wisc.edu/fri/jyu.htm
Jurgen WENDLAND
Yeast Biology                                        Oscar ZARAGOZA
Carlsberg Laboratory                                 Centro Nacional de MicrobiologÌa, Servicio de
Gamle Carlsberg Vej 10                               MicologÌa
Copenhagen 2000, Denmark                             Instituto de Salud Carlos III
Tel : 4533275 230                                    Carretera Majadahona-Pozuelo, Km 2
Fax : 4533274 708                                    Majadahonda, Madrid 28220, Spain
Mail: jww@crc.dk                                     Tel : +34 91 822 3661
                                                     Fax : + 34 91 509 7034
Ted WHITE                                            Mail: ozaragoza@isciii.es
Pathobiology                                         Web :
Seattle Biomedical Research Institute                http://www.isciii.es/htdocs/centros/microbiolo
307 Westlake Ave N Ste. 500                          gia/servicios/micro_servicio_micologia.jsp
Seattle WA 98109-5219, United States
Tel : 12062567 344                                   Martin ZAVREL
Fax : 1 206 256-7229                                 MBT
Mail: white@sbri.org                                 Fraunhofer IGB
                                                     Nobelstrasse 12
Malcolm WHITEWAY                                     Stuttgart 70569, Germany
Health Sector                                        Tel : +49 (0)711 970 4048
NRC Biotechnology Research Institute                 Fax : +49 (0)711 970 4200
6100 Royalmount Ave.                                 Mail: zav@igb.fraunhofer.de
Montreal QC H4P 2R2, Canada
Tel : 15144966 146                                   Ute ZEIDLER
Fax : 15144966 213                                   Department of Cell Biology
Mail: malcolm.whiteway@cnrc-nrc.gc.ca                University of Salzburg
                                                     Hellbrunnerstrasse 34
Duncan WILSON                                        Salzburg 5020, Austria
Microbial Pathogenicity Mechanisms                   Tel : 4366280445 793
Hans Knˆll Institut                                  Fax : 436628044 144
Beutenbergstrasse                                    Mail: ute.zeidler@sbg.ac.at
Jena D-07745, Germany
Tel : +49 (0) 3641 65 68 85
Fax : +49 (0) 3641 65 68 82
Mail: Duncan.Wilson@hki-jena.de
Web : http://www2.hki-jena.de/rz/hki_i00.htm

Tim YEOMANS
Division of Oral Biosciences
Dublin Dental School and Hospital
Lincoln Place
Dublin 2, Ireland
Tel : 00353(0)16127366
Fax : 00353(0)16127297
Mail: tim.yeomans@dental.tcd.ie


                                               227
                           INDEX OF AUTHORS
                           Bignell, E.: S15, P19,       Castillo, L.: P36, P105
Abed, U.: P143             H22, P127                    Castro, A.: P138
Abrunheiro, A.: P94        Binkley, G.: P80             Castro, G.: P121
Adamczak, R.: S11          Bistoni, F.: P150            Cenci, E.: P150
Alcazar Fuoli, L.: P100    Bito, A.: P4                 Cerutti, L.: P78
Aliouat, C.: P3            Blafl-Warmuth, J.: P79        Chabe, M.: P3
Aliouat, E.: P3            Blass-Warmuth, J.: P77       Chaloupka, J.: P33
Almeida Soares, C.: P115   Bolstad, M.: P24             Chen, J.: P37, P38
Almeida, A.: P32           Borges, C.: P115             Chorvat, D.: P73
Almo, S.: P139             Borth, N.: P45               Citiulo, F.: P35
Aloisi, T.: P138           Botero, S.: P123             Ciudad, T.: P9
Alvarez, J.: P55           Bourgeois, C.: P134,         Cockell, M.: P78
Amedeo, P.: S15            P141, P146                   Coleman, D.: P35, P42,
Ammerer, G.: P53           Brachhold, M.: P58, P59      P44, P54, P93, P120,
Andaluz, E.: P9            Brakhage, A.: P30, P106,     P130
Anderson, J.: S14          P107, P135, P136             Corbucci, C.: P150
Anjos, J.: P94             Breitenbach, M.: P4          Cormack, B.: S45
Arkowitz, R.: P63          Brinkmann, V.: P143          Correa-Bordes, J.: P15
Armstrong-James, D.:       Bromley, M.: P95             Correia, A.: P121
H22, P127                  Brown, A.: P1, P17, P22      Costa-de-Oliveira, S.: P96
Arnaud, M.: P80            Brown, G.: S53               Costanzo, M.: P80
Arnold, J.: S11            Brunke, S.: P113             Coste, A.: P69, P70
Arroyo, J.: S44            Brunner, H.: P81             Crabtree, J.: S15
Arst, H.: P19              Buck, J.: P33                Crielaard, W.: P43, P110
Atir-Lande, A.: P26, P52   Bujdakova, H.: P72, P73      Crittin, J.: P70
Aubert, S.: P88            Butler, G.: S12, P29, P56,   Cuenca Estrella, M.:
Aversa, F.: P138           P147                         P100, P117
Azeredo, J.: P18, P71,     Caballero-Lima, D.: P15      Cunha, C.: P32
P104                       Cabezon, V.: S44, P125       Cushion, M.: S11
Bader, O.: P83, P144       Calderon, J.: P98            Dabas, N.: P49
Baeza, L.: P115            Calderone, R.: P9            Dabkowska, M.: P8
Bai, Q.: P149              Cannon, R.: P66              Davidson, A.: P2
Bail„o, A.: P115           Cantero, P.: P5              De Bernardis, F.: P50
Bain, J.: P2               Cao, F.: P37                 de Boer, A.: P122
Barberat, A.: P19          Caplice, N.: P130            De Brucker, K.: P27
Barker, K.: P79, P84       Cardenas-Corona, M.:         de Groot, P.: P43, P122
Barreto, L.: P31           P34                          Decker, T.: P141
Bassilana, M.: P63         Carman, A.: P114             Degel, B.: P77
Bastidas, R.: P34          Carmona, J.: P32             Dei-Cas, E.: P3
Bauser, C.: P91            Carotti, A.: P138            Del Bel Cury, A.: P110
Beckett, R.: P12           Carr, P.: P95                del Castillo, L.: P105
Behnsen, J.: P135, P136    Carreto-Binaghi, L.: P3      d'Enfert, C.: S24, P64,
Berg, M.: P61              Carvalho, A.: P138           P88, P101
Berkes, C.: P142           Casadevall, A.: H12,         Deng, D.: P110
Berman, J.: S33, P69       P117                         Deutscher, E.: P26
Bermejo, C.: S44           Casas, C.: P31               Diez-Orejas, R.: P87
Beser, J.: P123            Cassone, A.: S35, P50        Díez-Orejas, R.: S44


                                      228
Ding, C.: P29              Gonçalves, T.: P94          Huang, G.: P38
Dini, L.: P89              Gonçalves-Rodrigues, A.:    Hube, B.: P112, P113,
Distel, B.: P28, P103      P96                         P144
Domergue, R.: S45          Gonzalez-Novo, A.: P15      Hull, C.: P10, S13, P126
Dostal, J.: P75, P90       Gow, N.: P2, P22, P23,      Husickova, V.: P41
Dromer, F.: S24            S32                         Hust, M.: P151
du Plessis, M.: P89        Goyard, S.: S24, P64,       Inglis, D.: P142
Dübel, S.: P151            P88, P101                   Iraqui, I.: S24, P88
Dukalska, M.: P59          Gregori, C.: P40, P53       Isaac, D.: P128
Dünkler, A.: P6            Gross, U.: P122             Ischer, F.: P69, P70
Eichholz, S.: P151         Guenther, M.: P119          Izquierdo, A.: P31
Ellis, D.: P2              Guerardel, Y.: S41          Jacobsen, M.: P2
Enjalbert, B.: P22         Gunzer, M.: P135, P136      Janbon, G.: S24, S41,
Ermert, D.: P143           Gutierrez-Escribano, P.:    S42, P64, P88, P101,
Ernst, J.: P5, P39, P150   P15                         P132
Fedorova, N.: S15, P127    Haas, H.: P107              Jimenez, A.: P145
Fernandez, V.: P89, P123   Hagblom, P.: P123           Jimenez, J.: P15
Fernández-Arenas, E.:      Hanby, R.: S22              Joardar, V.: S15
S44                        Hardy, G.: P103             Johnson, A.: S52, P133
Ferrari, S.: P68, P91      Hartmann, T.: P51           Jones, L.: P46
Filler, S.: S24, P76       Hasenberg, M.: P135,        Jouault, T.: S41
Fiori, A.: P57             P136                        Jungblut, P.: P143
Firon, A.: P88             Hauser, N.: P59, P118,      Kadzielska, J.: P8
Flegelova, H.: P75         P119                        Kallnischkies, M.: P62
Fleischhacker, M.: P60,    Hauser, P.: P78             Karstaedt, A.: P89
P124, P148                 Haynes, K.: H22, P127       Kawamoto, S.: P41
Foley, E.: S52             Heierhorst, J.: P16         Kawecki, D.: P8
Fontaine, T.: S42          Heinekamp, T.: P30          Kaye, S.: P95
Forche, A.: S33, P69       Heitman, J.: P10, P34,      Kegang Zhu, K.: P80
Fradin, C.: S41, P131,     P76                         Keller, N.: S43
P132                       Henriques, M.: P18, P71,    Kelly, M.: P116
Frean, J.: P89             P104                        Kenny, C.: P147
Freeman, G.: P139          Hermosa, B.: P9             Kenya, M.: P66
Frohner, I.: P134, P141,   Hernandez, R.: P118,        Kibbler, C.: P2
P146                       P119                        Kim, S.: P127
Fung, E.: H21              Herrero, E.: P31            Kirsch, M.: P151
Gabaldon, T.: P13          Hillenmeyer, M.: H21        Klein, B.: S21
Gabrielli, E.: P150        Hiller, E.: P81             Klis, F.: P122
Gacser, A.: P108, P139     Hinz, D.: P151              Kniemeyer, O.: P106,
Garcera, A.: P31           Hnisz, D.: P25              P107
Garcia Effron, G.: P100    Hogues, H.: S25             Kofla, G.: P148
Gasperik, J.: P73          Hokamp, K.: P120            Kohn, L.: P133
Gebhart, D.: S22           Holmes, A.: P66             Kolecka, A.: P73
Gehrke, A.: P30            Homayouni, R.: P79, P84     Konopka, J.: P55
Giaever, G.: H21           Hoon, S.: H21               Kornitzer, D.: P26, P52
Gil, C.: S44, P87, P125,   Hoot, S.: S31, P86          Kraidlova, L.: P102
P145                       Hope, H.: P63               Kraneveld, E.: P43, P110
Gildor, T.: P26            Howsley, S.: P95            Krappmann, S.: P136
Giles, S.: P126            Hradilek, M.: P90           Krauke, Y.: P20
Glaser, W.: P21, P25       Hruskova-                   Kucharikova, S.: P72, P73
Goldman, G.: S23           Heidingsfeldova, O.: P75,
Gomez-Raja, J.: P9         P90

                                     229
Kuchler, K.: P21, P25,     Martins, M.: P18, P71       Nislow, C.: H21
P40, P53, P134, P141,      Mateus, D.: P7              Noble, S.: P133
P146                       McDonagh, A.: P127          Nombela, C.: S44, P87,
La Valle, R.: P50          McGirt, L.: P10             P125, P145
Laforet, L.: P36           Meinke, A.: P82             Nordheim, A.: P81
Lagrou, K.: P109           Meliao-Silvestre, A.: P94   Nosanchuk, J.: P108,
Lamping, E.: P66, P99      Mellado Terrado, E.:        P139
Lane, R.: P47              P100                        Novotny, R.: P41
Larriba, G.: P9            Meller, J.: S11             O'Connor, L.: P42
Laurent, C.: S24           Mendes Giannini, M.:        Odds, F.: P2, P22, P44
Lavoie, H.: S25            P115                        O'Farrell, P.: S52
Lazar-Molnar, E.: P139     Mille, C.: S41, P131,       O'Grimalt, J.: P100
Leao, C.: P32              P132                        Ohkusu, M.: P41
Lebbad, M.: P123           Miranda, I.: P7             Ohlsen, K.: P48
Lee, W.: H21               Misselwitz, S.: P39         Oliveira, R.: P18, P71,
Lengsfeld, C.: P5          Miyasato, S.: P80           P104
Lermann, U.: P77, P111     Mogensen, E.: P101          Oliver, B.: S31
Lessing, F.: P106          Mol, E.: P103               Oliver, J.: P95
Levin, L.: P33             Monk, B.: P66               Ophir, A.: P52
Levitz, S.: S55            Monod, M.: P77              Pais, C.: P121
Li, Y.: P37                Monteiro, J.: P94           Pais, H.: P7
Lin, Y.: P66               Monteoliva Diez, L.: P87    Palmer, G.: P116
Lindemann, E.: P11, P61,   Moran, G.: P35, P42, P44,   Pan, S.: S45
P62                        P54, P93, P120, P130        Park, H.: S24
Lisalova, M.: P72          Moranova, Z.: P41           Park, Y.: P48
Liu, T.: P79, P84          Moreno, I.: P36, P105       Pasligh, J.: P148
Lo Presti, L.: P78         Morschhäuser, J.: P48,      Pasrija, R.: P67
Logue, M.: S12, P147       P49, P77, P79, P84, P108,   Pavlicek, J.: P41
Lopez, J.: P100            P111                        Pavlickova, L.: P90
Lorenz, M.: P114           Mosci, P.: P150             Pedrosa, J.: P121
Loss, O.: P19              Moyes, D.: P149             Pereira, M.: P115
Luczak, M.: P8             Moyrand, F.: S42            Pereira, T.: P43, P110
Ludovico, P.: P32          Mtshali, N.: P12            Pichova, I.: P75, P90
Lunderius, C.: P140        Mühlschlegel, F.: P24,      Piedrafita, L.: P31
Ma, B.: S45                P33                         Piekarska, K.: P103
MacCallum, D.: P44         Mulhern, S.: P56            Pierce, S.: H21
Macedo-Ribeiro, S.: P7     Munier, H.: P64             Pina-Vaz, C.: P96
Maciel, P.: P138           Munier-Lehman, H.: S24      Pinheiro, M.: P7
MacrÏ, C.: P50             Munro, C.: S32              Pinjon, E.: P93
Mah, J.: P14               Naglik, J.: P149            Pitarch, A.: P145
Maidan, M.: P102           Nagy, E.: P82               Pitzurra, L.: P138
Maiti, R.: S15             Nantel, A.: S25             Pla, J.: P5
Majer, O.: P134, P141,     Narang, P.: P135, P136      Popolo, L.: P98
P146                       Nathenson, S.: P139         Porollo, A.: S11
Majewska, A.: P8           Naulleau, C.: S24, P64      Poulain, D.: S41, P131,
Manders, E.: P110          Nguyen, V.: S22             P132
Mangos, M.: S25            Nicholls, S.: P17           Prasad, R.: P67
Mannan, A.: P33            Nierman, W.: S15, P127      Prill, S.: P5
Mao, X.: P37               Niimi, K.: P66              Proctor, M.: H21
Martchenko, M.: S25        Niimi, M.: P99              Raclavsky, V.: P41
Martinez, A.: P36, P105    Nikolaou, E.: P1            Radecke, C.: P148
Martinez-Lopez, R.: P87    Nilsson, G.: P140           Ramirez, M.: P114

                                     230
Raymond, M.: P84            Schüller, C.: P40, P53      Sullivan, D.: P35, P42,
Reedy, J.: P10, P76         Schulz, B.: P60, P124       P44, P54, P93, P120,
Regenbogen, J.: P11, P91    Schütte, M.: P151           P130
Reuss, O.: P48              Schwartz, P.: S24           Svirshchevskaya, E.:
Ribeiro, A.: P71            Schwarzmueller, T.: P21,    P137
Ricardo, E.: P96            P25                         Swoboda-Kopec, E.: P8
Richards, A.: P23           Selander, C.: P140          Sychrova, H.: P20, P75,
Richards, T.: S31           Sellam, A.: S25             P102
Ricicova, M.: P109          Selmecki, A.: S33, P69      Synnott, J.: P147
Riera, M.: P101             Sentandreu, R.: P36, P105   Takano, Y.: P99
Rocha, R.: P7               Sesterhenn, T.: S11         Tanabe, K.: P99
Rodaki, A.: P22             Shah, P.: P80               Tavanti, A.: P2
Rodrigues, F.: P32, P138    Shaw, D.: P2                Taylor, M.: P3
Rodriguez Tudela, J.:       Shekhovtsova, E.: P137      ten Cate, B.: P43, P110
P100                        Sherlock, G.: P80           Tomiyama, S.: P99
RodrÌguez Tudela, J.:       Shevchenko, M.: P137        Traven, A.: P16
P117                        Sil, A.: S22, P128, P142    Trinel, P.: S41, P131,
Rodriguez-Arellanes, G.:    Silva, S.: P104             P132
P3                          Silva-Dias, A.: P96         Trkulja, D.: P62
Roehm, M.: P62              Silver, P.: S31             Trofa, D.: P108
Roetzer, A.: P40, P53       Simanis, V.: P78            Tuckwell, D.: P95
Rogers, P.: P79, P84        Skrzypek, F.: P150          Turner, G.: S15
Rohde, B.: P11, P91         Skrzypek, M.: P80           Uehara, Y.: P99
Rolli, E.: P98              Slaven, B.: S11             Urban, C.: P58, P62, P143
Roman, E.: P5               Smulian, A.: S11            Valentin, E.: P36, P105
Romani, L.: P138            Snoep, J.: H23              van den Berg, M.: P103
Routier, F.: P136           Sohn, K.: P11, P61, P62,    van den Burg, J.: P103
Ruhnke, M.: H11, P60,       P118, P119                  Van Dijck, P.: P27, P74,
P124, P148                  Sohr, R.: P60               P102, P109
Rupp, S.: P11, S34, P58,    Solis, N.: S24              van Roermund, C.: P103
P59, P61, P62, P81, P118,   Somai, B.: P152             van Winkelhoff, A.: P43
P119, P134                  Song, J.: S31               Vazquez de Aldana, C.:
Rustchenko, E.: P9          Spiering, M.: P120          P15
Samaniego, C.: S31, P85     St. Onge, R.: H21           Vecchiarelli, A.: S54,
Sampaio, P.: P121           Staben, C.: S11             P150
Sampaio-Marques, B.:        Staib, P.: P77              Veses, V.: P23
P32                         Stanton, B.: S13            Vi, S.: P65
Sandini, S.: P50            Stateva, L.: P57            Vinterova, Z.: P75
Sanglard, D.: P68, P69,     Staudt, M.: S13             Visser, W.: P28
P70                         Stelmach, E.: P8            von Gabain, A.: P82
Santos, M.: P7, P121        Stockinger, S.: P141        Walker, L.: S32
Schäfer, W.: P108           Strijbis, K.: P28, P103     Walther, A.: P45
Schaller, M.: P122, P149    Stroschein-Stevenson, S.:   Wang, H.: P38
Schaub, Y.: P45             S52                         Weber, K.: P60, P124
Scheynius, A.: S51, P140    Stumpf, M.: H22             Weig, M.: P83, P122
Schirmeister, T.: P77       Sturtevant, J.: P116        Weindl, G.: P122, P149
Schirrmann, T.: P151        Stynen, B.: P74             Wendland, J.: P6, P45
Schlick, P.: P82            Su, C.: P37                 White, T.: S31, P85, P86
Schmalhorst, P.: P136       Subanovic, M.: P5           Whiteway, M.: S25
Schmid, M.: P143            Sudbery, P.: P47, P65       Williams, D.: P104
Schmidt, A.: P107           Suleman, E.: P152           Wilson, D.: P112
Schmidt, I.: P39            Sulik-Tyszka, B.: P8        Wong, M.: P89

                                      231
Wortman, J.: S15
Würzner, R.: P77
Xiong, X.: P58
Xu, L.: P84
Yeomans, T.: P54
Yu, J.-H.: P14
Yu, Y.: P127
Zaragoza, O.: P117
Zavrel, M.: P118
Zeidler, U.: P4
Zelt, G.: P119
Zhou, H.: P114
Zimmermannova, O.: P20
Znaidi, S.: P84
Zupancic, M.: S45
Zychlinsky, A.: P143




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