Human Fungal Pathogens May 2-8_

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Human Fungal Pathogens May 2-8_ Powered By Docstoc
					Third FEBS Advanced Lecture Course
  Human Fungal Pathogens
 Molecular Mechanisms of Host-Pathogen
        Interactions and Virulence
          May 2-8, 2009
      La Colle sur Loup, France



      Genomics, evolution and epidemiology
   Environmental sensing and morphogenesis
Antifungal strategies and Mechanisms of resistance
            Host-pathogen interactions
        Systems Biology in pathogenesis



                    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)
                  Arturo Casadevall (USA)
                   Melanie Cushion (USA)
                  Patrick van Dijck (Belgium)
                    Cameron Douglas (USA)
                  Paul Dyer (United Kingdom)
                        Scott Filler (USA)
                  Gustavo Goldman (Brazil)
                Ken Haynes (United Kingdom)
                   Joseph Heitman (USA)
                     Nancy Keller (USA)
              Joachim Morschhäuser (Germany)
               Carol Munro (United Kingdom)
                Mihai Netea (The Netherlands)
                 Annika Scheynius (Sweden)
                   Derek Sullivan (Ireland)
              Hanna Sychrova (Czech Republic)
                    Yue Wang (Singapore)


                           2
                   Sponsors

The Federation of European Biochemical Societies




                       3
       Important informations                                                    p. 4

       Indicative time table                                                     p.

       Program                                                                   p.

       Lecture abstracts                                                         p.

       Posters and workshop talks abstracts                                      p.

       List of participants                                                      p.

       Index of authors                                                          p.



                              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 2 and May 3 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
Sunday May 3 before the session starts and removed Thursday May 7.
Participants should be at their poster on the following day:
Sunday May 3: Posters with the letter A
Monday May 4: Posters with the letter B
Wednesday May 6: 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 2 – May 7 inclusive (6 nights), with departure
after breakfast on Friday May 8. All participants will stay in the Club Belambra 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 Club Belambra 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 Club Belambra 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 Club Belambra 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 15 to 20 °C during the day and 8 to 12°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 5.

Social Programme
A welcome drink will take place on Saturday May 2 and a special conference dinner and aperitif
will be served on the evening of Thursday May 7.

Excursions will be proposed during the free afternoon of May 5. Four 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
The evening of May 5 will be free. Buses will be organized to return to the site from different
locations where the participants could have dinner.


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 Club Belambra. 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
Club Belambra.




                                                7
8
INDICATIVE TIME-TABLE




          9
                           PROGRAM

                        Saturday May 2, 2009
3.00 pm     Registration

7.00 pm     Dinner

8.30 pm     Keynote lecture

  8.30 pm   Christophe d’Enfert, Opening of the meeting

  8.40 pm   Joe Heitman, Duke University, USA
            Microbial pathogens in the fungal kingdom

  9.30 pm   Welcome drinks



                           Sunday May 3, 2009
7.30 am     Breakfast

8.00 am     Registration

9.00 am     Session 1: Pathogenic fungi - genomics, evolution and epidemiology
            Chair: Melanie Cushion

  9.00 am   Melanie Cushion, University of Cincinatti, USA
            Introduction

  9.20 am   Frank Odds, University of Aberdeen, United Kingdom
            Epidemiology of Candida albicans infections

  9.50 am   Derek Sullivan, Trinity College Dublin, Ireland
            Comparative analysis of the genomes and transcriptomes of Candida
            albicans and Candida dubliniensis

  10.20 am Coffee break

  10.40 am Paul Dyer, Nottingham University, United Kingdom
           Clandestine Sexual Activity in Fungal Pathogens


  11.10 am John Taylor, University of California at Berkeley, USA
           Genomics of adaptation: Coccidioides and related Ascomycota



                                    10
  11.40 am Patrick Keeling, University of British Columbia, Vancouver, Canada
           Evolution of Microsporidia

12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 1: Pathogenic fungi - genomics, evolution and epidemiology
            Chairs : Paul Dyer and Patrick van Dijck

  4.00 pm   Manuel Santos, University of Aveiro, Portugal
            Crystal structures of the SerRS explain proteome tolerance to a genetic
            code alteration in Candida albicans

  4.20 pm   Kai Sohn, Fraunhofer IGB, Germany
            Open platform technologies for unbiased analyses of gene expression in
            fungal pathogens

  4.40 pm   Yeissa Chabrier-Rosello, University of Rochester School of Medicine &
            Dentistry, USA
            Large scale synthetic genetic analysis of the RAM signaling network in
            C. albicans

  5.00 pm   Marina Marcet-Houben, Center for Genomic Regulation, Spain
            Fungal phylogenomics

  5.15 pm   Tobias Schwarzmüller, Medical University of Vienna, Austria
            A systematic approach to identify virulence and drug resistance genes
            in the human fungal pathogen Candida glabrata

  5.30 pm   Denise Lynch, University College Dublin, Ireland
            G+C content variation in the Saccharomycotina

  5.45 pm   Amanda Gibson, Université Paris-Sud XI, France
            Hybridization and phylogenetics in predicting pathogen emergence on a
            novel host

7.00 pm     Dinner

8.30 pm     Poster session



                        Monday May 4, 2009
7.30 am     Breakfast

9.00 am     Session 2: Environmental sensing and morphogenesis




                                    11
            Chair: Geraldine Butler

  9.00 am   Geraldine Butler, University College Dublin, Ireland
            Introduction

  9.20 am   Yue Wang, Institute of Molecular and Cell Biology, Singapore
            Candida albicans hyphal development---from signal sensing to polarity
            control

  9.50 am   Fritz Mühlschlegel, University of Kent, Canterbury, United Kingdom
            Environmental sensing in the fungal pathogen Candida albicans

  10.20 am Coffee break

  10.40 am Elaine Bignell, Imperial College, London, United Kingdom
           Molecular modelling of A. fumigatus signal reception in response to
           environmental shift

  11.10 am Alex Andrianopoulos, University of Melbourne, Victoria, Australia
           Control of morphogenesis and responses to the host by Penicillium
           marneffei

  11.40 am Alex Idnurm, University of Missouri-Kansas City, USA
           Light-sensing and its impact on virulence in pathogenic fungi

12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 2: Environmental sensing and morphogenesis
            Chairs : Al Brown and Yue Wang

  4.00 pm   Christoph Schüller, Medical University of Vienna, Austria
            The metabolic response of Candida glabrata to phagocytosis

  4.20 pm   Jaime Correa-Bordes, Universidad de Extremadura, Spain
            Regulation of septin dynamics through Rts1 during Candida albicans
            morphogenesis

  4.40 pm   Christian Schmauch, Université Nice-Sophia Antipolis, France
            Systematic analysis of kinase and phosphatase function in Candida
            albicans‘ yeast to hyphae transition

  5.00 pm   Patricia Albuquerque, Albert Einstein College of Medicine, USA
            Cell density regulation of growth, GXM release and melanization in
            Cryptococcus neoformans

  5.15 pm   Michelle Leach, Aberdeen University, United Kingdom
            The conservation of a heat shock response in an obligate pathogen of
            warm-blooded animals


                                     12
  5.30 pm    Chang Su, State Key Laboratory of Molecular Biology, China
             Mss11, a transcription activator, is required for hyphal development in
             Candida albicans

  5.45 pm    Audrey Nesseir, Institut Pasteur, France
             Understanding the role of the Candida albicans Yak1 kinase in the
             regulation of hyphal growth

7.00 pm      Dinner

8.30 pm      Poster session



                              Tuesday May 5, 2009
7.30 am      Breakfast

9.00 am      Session 3: Antifungal strategies and mechanisms of resistance
             Chair: Carol Munro

  9.00 am    Carol Munro, University of Aberdeen, United Kingdom
             Introduction

  9.20 am    Joachim Morschhäuser, Würzburg Universität, Germany
             Transcriptional control of drug resistance in Candida albicans

  9.50 am    Jean-Paul Latgé, Institut Pasteur, Paris, France
             The cell wall of A. fumigatus

  10.20 am Coffee break

  10.40 am Aaron Mitchell, Carnegie Mellon University, Pittsburgh, USA
           Biofilm matrix regulation by Candida albicans Zap1

  11.10 am Antonio Cassone, Istituto Superior di sanita, Roma, Italy
           Fungal vaccines: a critical view

  11.40 am Terry Roemer, Merck Frosst Canada, Montreal, Canada
           Fungal genomics and natural product discovery: in search of novel
           antifungal agents

12.15 pm     Lunch

1.30 pm      Free afternoon and dinner




                                         13
                        Wednesday May 6, 2009
7.30 am     Breakfast

9.00 am     Session 4: Host-pathogen interactions
            Chair: Bernhard Hube

  9.00 am   Bernhard Hube, Hans Knoell Institute, Germany
            Introduction

  9.20 am   Axel Brakhage, Hans Knoell Institute, Jena, Germany
            Mechanisms of the interaction of Aspergillus fumigatus with immune
            effector cells

  9.50 am   Bill Goldman, University of North Carolina, Chapel Hill, USA
            Phase-specific genes and intracellular survival strategies of Histoplasma
            capsulatum

  10.20 am Coffee break

  10.40 am Scott Filler, Harbor UCLA Medical Center, Torrance, USA
           Mechanisms of invasion of host cells by Candida albicans

  11.10 am Dominique Ferrandon, IBMC-CNRS, Strasbourg, France
           Drosophila melanogaster as a model to study host-fungi interactions

  11.40 am Mihai Netea, Radboud University, Nijmegen, The Netherlands
           Host defense against Candida albicans infections: from Drosophila to
           the patient

12.15 pm    Lunch

3.30 pm     Coffee and tea

4.00 pm     Workshop 3: Antifungal strategies and mechanisms of resistance
            Chairs : Joachim Morschhäuser and Derek Sullivan

  4.00 pm   André Nantel, Biotechnology Research Institute, Canada
            Genome-wide mapping of the coactivator ADA2 yields insight into the
            functional roles of SAGA/ADA complex in Candida albicans

  4.20 pm   Arnold Bito, University of Salzburg, Austria
            Role of the C. albicans ortholog of yeast GCS1 in multidrug resistance
            and hyphal growth

  4.40 pm   Sélène Ferrari, CHUV, Switzerland
            Gain of function mutations in CgPDR1 of C. glabrata not only mediate
            antifungal resistance but also enhance virulence


                                    14
  5.00 pm   Raymond Rowan, Dublin Dental School & Hospital, Ireland
            Investigations into the anti-C. albicans activity of a synthetic
            decapeptide with yeast killer toxin like activity

  5.20 pm   Amandine Gastebois, Institut Pasteur, France
            Characterisation of the first cell wall beta(1-3)glucan branching activity
            in Aspergillus fumigatus


  5.35 pm   Elias Epp, McGill University, Canada
            Reverse genetics in the human fungal pathogen C. albicans aiming at
            improving current drug treatment options

  5.50 pm   Michaela Mai, Fraunhofer IGB, Germany
            Development of a universal system for fungal species identification and
            SNP typing via on-chip minisequencing

7.00 pm     Dinner


                        Thursday May 7, 2009

7.30 am     Breakfast

9.30 am     Session 5: Systems biology in pathogenesis
            Chair: Judith Berman

  9.30 am   Judith Berman, University of Minnesota, Minneapolis, USA
            Introduction

  9.50 am   Tim Galitski, Institute for Systems Biology, Seattle, USA
            Systems genetics of yeast filamentation

  10.20 am Brenda Andrews, University of Toronto, Canada
           Deciphering cellular networks and pathways using yeast functional
           genomics

  10.50 am Coffee break

  11.10 am Ken Haynes, Imperial College, London, United Kingdom
           Functional genomics of fluconazole sensitivity in Candida glabrata using
           a library of transcription factor knock-outs

  11.40 am Thomas Höfer, German Cancer Research Center, Heidelber, Germany
           Molecular networks of T-helper cell differentiation: from
           experiments to computational models and back

12.15 pm    Lunch


                                      15
3.00 pm        Coffee and tea

3.30 pm        Workshop 4: Host-pathogen interactions
               Chairs : Scott Filler and Joe Heitman

  3.30 pm      Constantin Urban, Umea University, Sweden
               Neutrophil extracellular trap formation releases S100 proteins crucial
               for antifungal immune responses

  3.50 pm      Oscar Zaragoza, ISCIII, Spain
               Fungal gigantism during mammalian infection

  4.10 pm      Annika Scheynius, Karolinska Institute, Sweden
               TLR2/MyD88-dependent and -independent activation of mast cell IgE
               responses by the skin commensal yeast Malassezia sympodialis

  4.30 pm      Lucy Holcombe, University College Cork, Ireland
               Candida glabrata infection: alternative host models and the role of
               calcium signalling

  4.50 pm      Timothy Cairns, Imperial College, United Kingdom
               Stage specific gene expression profiling during initiation of invasive
               aspergillosis

  5.05 pm      Kerstin Voelz, University of Birmingham, United Kingdom
               Cytokine signaling regulates the outcome of intracellular macrophage
               parasitism by Cryptococcus neoformans

  5.20 pm      Olivia Majer, Medical University of Vienna, Austria
               Impact of Type I interferons on the cell-mediated immunity to Candida
               infection

5.35 pm        Anita Sil, concluding remarks

5.45 pm        Closing lecture
               Nick Talbot, Exeter University, United Kingdom
               Investigating the biology of plant infection by phytopathogenic fungi:
               lessons for the study of human pathogens

7.00 pm        Dinner and farewell party




                            Friday May 8, 2009
From 7.30 am          Breakfast and departure



                                           16
ABSTRACTS

LECTURES




    17
KEY-NOTE LECTURES




       18
S01
Microbial pathogens in the fungal kingdom
Joseph Heitman
Molecular Genetics and Microbiology, Duke University Medical Center, Research Drive, 322
CARL Building, Box 3546, Durham NC 27710, United States, Phone: 919 684-2824, FAX: 919
684-5458, e-mail: heitm001@duke.edu, Web:
http://www.mgm.duke.edu/microbial/mycology/heitman/

The fungal kingdom is vast and successful, spanning ~1.5 million species as diverse as unicellular
yeasts, filamentous fungi, mushrooms, lichens, and pathogens of both plants and animals. The
fungi are closely aligned with animals in one of the eight groups of eukaryotic organisms, the
opisthokonts. The two groups last shared a common ancestor ~1 billion years ago, much more
recently than with other groups of eukaryotic organisms. As a consequence of their close
evolutionary history and shared cellular machinery with metazoans, fungi are exceptional models
for mammalian biology, but thus prove to be difficult to treat in infected animals. The last
common ancestor to the fungal and metazoan lineages is thought to have been unicellular, aquatic,
and motile with a posterior flagellum, and certain extant species closely resemble this
hypothesized ancestor. Species within the fungal kingdom have traditionally been assigned to four
phyla, including the basal fungi (chytridiomycetes, zygomycetes) and the more recently derived
monophyletic lineage, the dikarya (ascomycetes, basidiomycetes). The recent fungal tree of life
project has revealed that the basal lineages are polyphyletic, and thus there may be as many as
eight fungal phyla. Fungi that infect vertebrates are found in all of the major lineages, and
virulence has arisen multiple times independently. A sobering recent development is that the
species Batrachochytrium dendrobatidis from one of the most basal phyla of the fungal kingdom,
the chytridiomycetes, has emerged to cause global amphibian declines and extinctions. Genomics
is revolutionizing our view of the fungal kingdom, and recent genome sequences for zygomycete
pathogens (Rhizopus, Mucor), fungi associated with the skin (dermatophytes, Malassezia), and the
Candida pathogenic species clade promise to provide considerable insights into the origins of
virulence. In this lecture, we will survey the diversity of fungal pathogens, and illustrate key
principles revealed by genomics involving sexual reproduction and sex determination, loss of
conserved pathways in derived fungal lineages that are retained in basal fungi, and shared and
divergent virulence strategies of successful human pathogens, including dimorphic and trimorphic
transitions in form.




                                               19
S02
Investigating the biology of plant infection by phytopathogenic fungi: lessons for the study of
human pathogens
Nicholas Talbot
Biosciences, University of Exeter, Geoffrey Pope Building, Exeter EX4 4QD, United Kingdom,
Phone: +44 1392 269151, FAX: +44 1392 263434, e-mail: n.j.talbot@exeter.ac.uk, Web:
http://cogeme.ex.ac.uk/talbot

Plant pathogenic fungi share many common features with human pathogenic species, including
exhibiting specific developmental processes associated with pathogenesis. The availability of
genome sequences for a wide variety of pathogenic and free-living fungal species has provided the
means to study the evolution of fungal pathogenicity and to define the genetic determinants
required to be a successful pathogen. The use of genome information has also provided the means
to investigate fungal-plant interactions in much greater detail than was hitherto possible and to
develop tools to rapidly evaluate gene function, allowing a systems biology approach to
understanding plant disease. We are studying rice blast disease caused by the ascomycete fungus
Magnaporthe oryzae, one of the most serious economic problems affecting rice production.
During plant infection, M. oryzae develops a differentiated infection structure called an
appressorium. This unicellular, dome-shaped structure generates cellular turgor, that is translated
into mechanical force to cause rupture of the rice cuticle and entry into plant tissue. My research
group is interested in determining the molecular basis of appressorium development and
understanding the genetic regulation of the infection process by the rice blast fungus. We have
recently shown that development of a functional appressorium is linked to the control of cell
division and autophagic programmed cell death in M. oryzae. Appressorium formation also
requires an oxidative burst that involving the action of NADPH oxidases and cellular
differentiation is coupled to an alteration in fungal metabolism leading to enormous turgor
generation in the infection cell. Once inside the plant, M. oryzae has evolved the ability to secrete
specific effector proteins into plant cells to suppress plant defences and allow invasion of living
plant tissue. Infection-related development, effector protein production and delivery are all
attributes shared with human pathogenic fungal species. Progress in these research areas and its
relation to human pathogenic species will be presented.
Reference:
Wilson, R.A. and Talbot, N.J. (2009) Under pressure: investigating the biology of plant infection
by the rice blast fungus Magnaporthe oryzae. Nature Reviews Microbiology 7: 185-195.




                                                 20
              SESSION 1

          PATHOGENIC FUNGI:
GENOMICS, EVOLUTION AND EPIDEMIOLOGY




                 21
S11
Epidemiology of Candida albicans infections
Frank Odds
Aberdeen Fungal Group, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK,
Phone: +44 (0) 1224 555828, FAX: +44 no fax no. use email, e-mail: f.odds@abdn.ac.uk

Candidaemia is a life-threatening condition that arises in a wide diversity of patient types whose
host anti-microbial defences are seriously impaired. While heightened clinical awareness and
prophylactic use of antifungal agents have halted the once steep rise in incidence of candidaemia,
the infection remains a difficult problem in clinical management.
Molecular methods for typing isolates of the main opportunistically pathogenic Candida species
have provided information on their epidemiology, evolution and phylogenetics. For C. albicans in
particular, multi-locus sequence typing (MLST) has generated a lot of data relating to commensal
and pathogenic behaviour of the fungus. MLST has shown intra-family transmission of C.
albicans isolates, but also that nosocomial transmission within hospitals is rare. The majority of C.
albicans isolates can be assigned to one of five major clades of closely related strain types. Each
clade, with the exception of the most common, clade 1, shows a geographical affinity suggestive
of a separate evolutionary path. Similarly, isolates from animals, particularly those from birds,
seem to belong to groups different from, but not yet entirely separated from, the types found in
humans.
The clades show significant differences in some properties, notably in prevalence and mechanism
of resistance to flucytosine, and in numbers of mid-repeat sequences in genes encoding surface
proteins, but examples of strains of high and low virulence in mouse models of candidaemia are
found in the all four of the largest clades.
Current technologies are moving towards development of micro-array based systems for
identification and typing of Candida spp. These promise to enhance laboratory support for
management of patients at high risk of invasive Candida infections.




                                                 22
S012
Comparative analysis of the genomes and transcriptomes of Candida albicans and Candida
dubliniensis
Derek Sullivan, Gary Moran and David Coleman
School of Dental Science, Trinity College Dublin, Lincoln Place, Dublin 2, IRELAND, Phone:
+353 (0)1 612 7275, FAX: +353 (0)1 612 7295, e-mail: Derek.Sullivan@dental.tcd.ie, Web:
http://people.tcd.ie/djsullvn

Candida dubliniensis is the species that is most closely related to Candida albicans, the most
pathogenic member of the genus Candida. However, despite their very close relationship,
epidemiological and infection model data show that C. dubliniensis is significantly less pathogenic
than C. albicans. In order to investigate the molecular basis of the differential virulence of the two
species we have compared their genomes. Comparative genomic hybridization experiments in
which genomic DNA from the two species was hybridized to C. albicans microarrays revealed
that approx. 96% of genes share a high level of homology. However, 247 genes were found to be
either absent or highly divergent in the C. dubliniensis genome, including genes encoding putative
virulence factors (e.g. HWP1, SAP4 and SAP6). These data have since been refined by the
completion of the C. dubliniensis genome by the Wellcome Trust Sanger Institute (see
http://www.sanger.ac.uk/sequencing/Candida/dubliniensis/). As expected, comparison of the C.
albicans and C. dubliniensis genomes revealed that they are highly similar and that synteny is
largely conserved. However, there are significant differences in the composition of a number of
gene families. Some of these have been previously associated with virulence (e.g. the SAP and
ALS families), while others have no known function (e.g. the telomere-associated TLO family and
the IFA family of putative transmembrane proteins). In addition to the absence of genes, C.
dubliniensis also possesses a larger number of pseudogenes than C. albicans, including several
filamentous growth regulator (FGR) genes that may play a role in morphogenesis. These data
suggest that C. albicans has undergone expansions of specific gene families while C. dubliniensis
may have also undergone reductive evolution of redundant loci following restriction to a
specialized, and as yet unidentified, ecological niche. Comparative transcriptomic analysis also
suggests that differential expression of specific genes is likely to contribute to phenotypic
differences between the two species. It is hoped that further dissection of the genomic differences
between these two species will identify novel virulence factors and improve our understanding of
the evolution of virulence in Candida spp.




                                                 23
S13
Clandestine Sexual Activity in Fungal Pathogens
Paul Dyer1, Céline O'Gorman2, Mathieu Paoletti1 and Hubert Fuller2
1 School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK,
Phone: +44 (0)115 9513203, FAX: +44 (0)115 9513251, e-mail: paul.dyer@nottingham.ac.uk,
Web: http://www.nottingham.ac.uk/biology/contacts/dyer/
2 School of Biology and Environmental Sciences, University College Dublin, Belfield, Dublin 4,
IRELAND

Many human pathogens have long-been considered as ‘asexual’ organisms due to the fact that
sexual structures have never been observed in the wild or in attempted laboratory crosses. Various
evolutionary theories have been put forward to explain why an exclusively asexual lifestyle might
be advantageous to such pathogens. However, there is accumulating evidence to suggest that many
supposedly ‘asexual’ species do in fact have a latent potential for sexual reproduction and might
indeed be partaking in clandestine sexual activity in nature. Evidence comes from genomic
analyses, studies of mating-type distribution and assessment of recombination rates in natural
populations. The presence of a functional sexual cycle is of great significance to the population
biology and evolution of a species, and would also provide a valuable genetic tool for classical
inheritance studies of traits of interest such as virulence and antifungal resistance. A series of
examples will be described from pathogenic Aspergillus, Penicillium and Tapesia (Oculimacula)
species where sexuality appears to be present in supposedly asexual species. Particular focus will
be on the discovery of a complete sexual cycle in the opportunistic pathogen A. fumigatus, which
has lead to the naming of the teleomorphic state Neosartorya fumigata1. The finding that many
supposedly asexual pathogens appear to have all the necessary genetic ‘machinery’ for sex raises
questions such as why has sex not been observed in these species, and why do they appear more
fastidious compared to closely related sexual species?

1O’Gorman CM, Fuller HT, Dyer PS (2009). Nature 457: 471-474.




                                               24
S14
Genomics of adaptation: Coccidioides and related Ascomycota
John W. Taylor1, Thomas J. Sharpton1, Jason Stajich1, Emily Whiston1, Christopher Ellison1,
Chiung-yu Hung2 and Garry Cole2
1 Plant and Microbial Biology, University of California, Berkeley, 111 Koshland Hall, Berkeley
CA 94720-3102, USA, Phone: + 510 642 5366, FAX: + 510 642 4995, e-mail:
jtaylor@nature.berkeley.edu, Web: http://pmb.berkeley.edu/~taylor/
2 University of Texas, San Antonio

Although most Ascomycetes associate principally with plants, the dimorphic fungi Coccidioides
immitis and C. posadasii are primary pathogens of immunocompetent mammals, including
humans. Infection is a consequence of environmental exposure to Coccidiodies, which is believed
to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and
evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C.
posadasii, a close, non-pathogenic relative, Uncinocarpus reesii, and a more diverged pathogenic
fungus, Histoplasma capsulatum, were sequenced and compared to sequenced genomes of 13
more distantly related Ascomycota, principally Eurotiomycetes. Comparing the Coccidioides
genome sequences with those of related fungi across a range of evolutionary distances revealed
various levels of genome evolution. We found changes in gene family size, gene gains, gene
losses and gene retentions. We also detected the effects of positive natural selection. These
analyses facilitated an understanding of how Coccidioides evolved to associate with animals and
identified genes with a putative role in the evolution of pathogenicity. These analyses also
identified the need for additional sequences to take advantage of more sophisticated evolutionary
analyses. These sequences include even closer nonpathogenic relatives and additional individuals
of the populations of pathogenic fungi.




                                               25
S15
Evolution of Microsporidia
Patrick Keeling
Botany, University of British Columbia, 6270 University Blvd., Vancouver BC V6T 1Z4, Canada,
Phone: 604 8224906, FAX: 604 8226089, e-mail: pkeeling@interchange.ubc.ca, Web:
www.botany.ubc.ca/keeling

The Microsporidia are a diverse group, with over 1,200 described species, composed entirely of
highly adapted, obligate intracellular parasites. All growth and division is intracellular, and outside
the host cell they only exist as spores. Spores are resistant, largely dormant cells dominated by a
complex infection apparatus. Spores are otherwise quite reduced, having lost or severely reduced
canonical structures such as mitochondria, peroxisomes, Golgi dictyostomes, or any 9 + 2
microtubular structures. Microsporidia are also reduced at most other levels, having little
metabolic diversity and among the smallest nuclear genomes known (as small as 2.3 Mbp). Their
genomes are highly reduced (few genes) and compacted (high gene density), and their genes
highly divergent, characteristics which have together led to some unusual developments in
genome dynamics and function. The extreme simplicity of microsporidian cells was once thought
to reflect their ancient, primitive nature, which was originally supported by molecular phylogeny.
However, for over a decade now evidence has accumulated that they are in fact related to fungi, a
conclusion now strongly supported. Whether microsporidia actually are fungi as opposed to being
a sister group to fungi as a whole remained unclear for some time, but both phylogenetic
reconstruction and more recently analyses of genome order conservation now both suggest they
emerge from within the fungi, probably closely related to zygomycetes. The realization that
microsporidia evolved from fungi transforms the way their unusual biology is interpreted – they
are no longer considered primitive, and are instead seen to be highly derived.




                                                  26
               SESSION 2

ENVIRONMENTAL SENSING AND MORPHOGENESIS




                  27
S21
Candida albicans hyphal development---from signal sensing to polarity control
Yue Wang
Genes and Development Division, Institute of molecular and Cell Biology, 61 Biopolis Drive,
Singapore 138673, Singapore, Phone: +65 65869521, FAX: +65 67791117, e-mail:
mcbwangy@imcb.a-star.edu.sg, Web: http://www.imcb.a-star.edu.sg/php/wy.php

The AIDS pandemic of the past 30 years has seen the rise of Candida albicans from a largely
benign commensal to the most prevalent fungal pathogens in humans. It often causes life-
threatening systemic infections in immunocompromised patients, leading to numerous deaths.
When C. albicans are exposed to the body fluid of the host, many genes are activated responsible
for a diverse range of infection- and virulence-related functions. One of the well accepted and
most extensively investigated virulence traits is C. albicans’ ability to switch growth forms
between yeast and hyphae. Therefore, elucidating the mechanisms that control the growth
transition and maintenance of each growth state is crucial for understanding the disease process
and developing new therapies. Furthermore, its polymorphism renders C. albicans an appropriate
model for addressing several fundamental biological issues such as polarity control and cell fate
determination. In my lecture, I will discuss recent discoveries of several key molecular events
along the entire pathway of hyphal development, including the nature of the hypha-inducing
signals in serum, the identity of the signal sensors, the adenylyl cylase Cdc35 as a signal
sensing/integration hub and it regulations, and molecular links between the upstream signaling
pathways and several key components of the polarity machinery.




                                               28
S22
Environmental sensing in the fungal pathogen Candida albicans
Rebecca Hall1, Fabien Cottier1, Clemens Steegborn2 and Fritz Muhlschlegel1
1 Biosciences, University of Kent, Giles lane, Canterbury CT27NJ, UK, Phone: +44 (0)1227
823988, FAX: +44 (0)1227 763912, e-mail: F.A.Muhlschlegel@kent.ac.uk, Web:
http://www.kent.ac.uk/bio/kfg/index.html
2 Department of Physiological Chemistry, Ruhr-University Bochum, Germany

C. albicans adapts to the diverse microbial habitats of its host. Mammalian environmental cues
including the body’s elevated CO2 levels and pH, the body’s elevated temperature and organic
molecules found in serum are all sensed by this fungus. C. albicans can respond to these host
signals by reversibly changing morphology between yeast and filamentous growth forms. This
transition is relevant for adhesion, biofilm formation and also enables the fungus to escape attack
from the immune system; hence it is considered an important virulence attribute. However, C.
albicans cells that form part of a growing fungal biomass, such as those found in superficial
epithelial infections or biofilms, may be exposed to signal gradients. Indeed, cells in the centre of
the fungal biomass will sense different conditions to those situated at the periphery, with volatile
signals being a predominant factor. Finally, many sites of the human body, including the
gastrointestinal tract and the skin, are co-inhabited by fungi and bacteria in mixed microbial
populations. Cell-density-sensing (quorum sensing) has been reported to have strong effects on C.
albicans morphology, with soluble mediators such as the sesquiterpene farnesol playing a critical
role. Cyr1p, encoding the C. albicans adenylyl cyclase, is essential for morphological
differentiation in response to many of the above-mentioned environmental cues. We have shown
that activation of recombinant Cyr1p can be mediated by elevated CO2/HCO3- concentrations, a
condition that C. albicans encounters in body niches, including blood (5.5% CO2/25mM
NaHCO3), pockets of the intestinal tract or the vaginal microhabitat. The fungal CO2-sensing
system includes another prominent enzyme, carbonic anhydrase, which catalyzes the formation of
bicarbonate and a proton from CO2 and water. The latter is also required for survival of C.
albicans under CO2-limiting conditions. In this talk I will give an introduction to C. albicans
environmental sensing, and present new findings on the molecular mechanism of adenylyl
cyclase-mediated CO2 sensing. I will also speak about the structure and regulation of the fungal
CO2-sensing system by using examples from our ongoing research in C. albicans, Cryptococcus
neoformans but also Saccharomyces cerevisiae. Finally, I will introduce the concept of self-
mediated gaseous communication as a form of CO2-socialisation in C. albicans.




                                                 29
S23
Molecular modelling of A. fumigatus signal reception in response to environmental shift
Elaine Bignell
Microbiology, Imperial College London, Armstrong Road, London SW7 2AZ, UK, Phone:
00442075942074, FAX: 00442075943095, e-mail: e.bignell@imperial.ac.uk, Web:
http://www1.imperial.ac.uk/medicine/about/divisions/is/microbiology/aspergillus/

Appropriate responses to environmental pH govern virulence of numerous fungal pathogens and
emerging experimental evidence reveals a complex interplay of transcription factor function
during alkaline adaptation. The proteolytically activated A. nidulans transcription factor, PacC, is
essential for growth at alkaline pH in vitro, a phenotype which extrapolates to severe attenuation
of virulence in neutropenic mice1. Other transcription factors which become important at high pH
in A. nidulans include the recently characterized SltA2 and calcium-responsive CrzA3 proteins,
both of which mediate cation tolerance, with differing ion-specificities, CrzA acts downstream of
the protein phosphatase, calcineurin, to regulate calcium tolerance in both A. nidulans and A.
fumigatus. An emerging picture of functions under control of these proteins offers insight on
normal responses to alkalinisation and ion stress, in particular, the molecular events occurring
downstream of transcription factor function.

In order to assess the physiological response of A. fumigatus to alkaline stress, and the role of
calcium signalling in such environmental adaptation, we have measured temporal gene expression
profiles following in vitro transfer from acidic to alkaline medium, and in response to calcium
exposure. Our analyses identify adaptation mechanisms of vastly different magnitude and
longevity. The datasets were analyzed independently and comparatively in order to identify stress-
specific responses and examine correlation between the two mechanisms, respectively.

Searching for molecular components upstream of transcription factors, we have scoured the
datasets for membrane components of alkaline adaptation. To identify molecular interactions
required for initiation of A. fumigatus alkaline adaptation we have also used the integral pH-
sensing plasma membrane protein PalH to isolate novel protein interactors, from a full length A.
fumigatus cDNA library, using the yeast membrane two hybrid (split-ubiquitin) system. Protein
interactions initiating A. fumigatus alkaline adaptation represent mechanisms non-essential for
fungal viability, but crucial for virulence, possibly presenting new opportunities to circumvent
infectious fungal growth.

1. Bignell, E., et. al. (2005) Mol Microbiol. 55:1072-1084.
2.Spielvogel A., et. al. 2008 Biochem J. 414:419-29
3. Soriani F., et. al. (2008) Mol. Micro. 67:1274-91




                                                30
S24
Control of morphogenesis and responses to the host by Penicillium marneffei
Kylie Boyce1, Sally Beard1, Alisha McLachlan1, David Canovas1, Lena Schrieder1, Luke Pase2,
Alicia Oshlack3, Gordon Smyth3, Natalie Federova4, Willaim Nierman4 and Alex
Andrianopoulos1
1 Department of Genetics, University of Melbourne, Royal Parade, University of Melbourne Vi
3010, Australia, Phone: 61 3 83445164, FAX: 61 3 83445139, e-mail: alex.a@unimelb.edu.au,
Web: http://www.genetics.unimelb.edu.au/research/andr/
2 Cancer & Haematology Division, Walter and Eliza Hall Institute
3 Bioinformatics Division, Walter and Eliza Hall Institute
4 J. Craig Venter Institute

Penicillium marneffei is an emerging fungal pathogen of humans, in particular those who are
immunocompromised. P. marneffei has the capacity to alternate between a hyphal and a yeast
growth form, a process known as dimorphic switching, in response to temperature. P. marneffei
grows in the hyphal form at 25°C and in the yeast form at 37°C. The hyphal form produces
conidia which are likely to be the infectious agent while the yeast growth form is the pathogenic
form found in infected patients. These yeast cells exist intracellularly in the mononuclear
phagocyte system of the host. The molecular events which establish and maintain the
developmental states and control of the dimorphic switching process in P. marneffei are poorly
understood.

Two genome-wide approaches have been taken to understand dimorphic switching and
pathogenicity. One approach was to sequence the genome of P. marneffei and its closest relative.
Comparison of the genomes of these two species has highlighted important differences between
them that may relate to pathogenicity. The other approach was to use a microarray-based
expression profiling approach to identify genes whose expression responded to changes between
the hyphal cells (grown at 25°C), yeast cells (grown at 37°C) and differentiated asexual
development cells (produced at 25°C). The microarray data identified numerous genes which
where morphological-type specific (hyphal cell, yeast cell or conidiophore cell) and well as cell-
type specific (unicellular and multicellular). Functional characterisation of a number of the
identified morphological-type specific genes has identified important pathogenicity determinants
and novel pathways controlling growth in the host.

This work is funded by National Health and Medical Research Council of Australia, National
Institute of Allergy and Infectious Disease and Howard Hughes Medical Institute.




                                               31
S25
Light-sensing and its impact on virulence in pathogenic fungi
Alexander Idnurm
School of Biological Sciences, University of Missouri-Kansas City, 5100 Rockhill Road, Kansas
City MO 64110, UNITED STATES, Phone: +1 816 235 2265, FAX: +1 816 235 1503, e-mail:
idnurma@umkc.edu, Web: http://sbs.umkc.edu/people/faculty/docIdnurmA.cfm

The majority of fungal species are hypothesized to sense and response to light. Light is a
ubiquitous environmental signal that provides information about the place of an organism in its
environment, the time of day, and yet light also represents a potential source of detrimental
ultraviolet radiation. A recently emerging area of microbial pathogenesis is that light-sensing is
required for virulence in both bacterial and fungal pathogens. In both groups, it is blue light
photoreceptors that regulate the ability to cause disease. In the fungi these are named White Collar
1 after the mutant phenotype of Neurospora crassa from which the gene was first cloned in 1996.
White Collar 1 homologs are required for virulence in the basidiomycete Cryptococcus
neoformans and ascomycete Fusarium oxysporum in mouse models of disease. In the
basidiomycete yeast C. neoformans, the light-sensing complex of Bwc1-Bwc2 also regulates
sexual reproduction and UV sensitivity that potentially impacts survival of the fungus in the wild.
While the Bwc1 and Bwc2 proteins have all the features for sensing light through a flavin-binding
domain, transmitting the signal between PAS domains in both proteins, and changing transcription
via a zinc finger DNA binding domain, the target genes for this complex are unknown. Two
complementary approaches to reveal these genes, which could include new virulence factors, have
been undertaken. Both microarray analysis of gene transcript abundance in response to light and
forward genetic screens reveal insights into the targets for photoregulation. In addition, C.
neoformans has a relatively simple photoresponse compared to other fungal species, and
represents an idea model for studying photoperception in the fungal kingdom.




                                                32
                     SESSION 3

ANTIFUNGAL STRATEGIES AND MECHANISMS OF RESISTANCE




                        33
S31
Transcriptional control of drug resistance in Candida albicans
Joachim Morschhäuser
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg D-
97070, Germany, Phone: +49 (0)931 312152, FAX: +49 (0)931 312578, e-mail:
joachim.morschhaeuser@mail.uni-wuerzburg.de, Web:           http://www.infektionsforschung.uni-
wuerzburg.de/research/mycology_unit/

Azole antifungal drugs, especially fluconazole, are widely used to treat infections caused by
Candida albicans, the most common human fungal pathogen. Azoles block the biosynthesis of
ergosterol, the main sterol in the fungal cell membrane, by inhibiting sterol 14alpha-demethylase,
which results in ergosterol depletion and production of toxic sterols. C. albicans can develop
resistance to azoles by various mechanisms, including alterations in the sterol biosynthesis
pathway that avoid the accumulation of toxic sterols, mutations in the target enzyme that reduce
its affinity for the drug, upregulation of ergosterol biosynthesis genes, and overexpression of
efflux pumps that actively transport azoles and other toxic substances out of the cell, resulting in
multidrug resistance. Three zinc cluster transcription factors play a central role in the regulation of
genes involved in drug resistance. Tac1 controls the expression of the ABC transporters CDR1
and CDR2, Mrr1 mediates the expression of the major facilitator MDR1, and Upc2 regulates the
expression of ergosterol biosynthesis genes. These transcription factors have additional target
genes that may also contribute to drug resistance. Gain-of-function mutations in each of the three
transcription factors result in constitutive upregulation of their target genes and increased drug
resistance. The aquisition of such mutations is frequently accompanied by loss of heterozygosity
and other genomic alterations, which further increases drug resistance of the strains. By combining
the various resistance mechanisms C. albicans can develop high-level, clinically relevant azole
resistance.




                                                  34
S32
The cell wall of A. fumigatus
Jean-Paul Latgé
Parasitology and Mycology, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, FRANCE,
Phone: +33 (0)1 40 61 35 18, FAX: +33 (0)1 40 61 341 9, e-mail: jean-paul.latge@pasteur.fr,
Web: http://www.pasteur.fr/recherche/unites/aspergillus/th1-aspergillus.htm

The cell wall of A. fumigatus is a unique structure which does not exist for human cells. It enables
the fungus to resist against external aggressions, but, at the same time, it is its Achilles’ heel since
it is a major drug target as shown by the commercial launch of echinocandins that block cell wall
biosynthesis.
Polysaccharides represent the major part of the fungal cell wall and are responsible for its rigidity
and plasticity. Six structural polysaccharides are present in the cell wall of A. fumigatus mycelium
and conidia: beta(1-3)glucan, chitin, galactomannan, beta(1-3/1-4)glucan, alpha(1-3)glucan and
galactosaminogalactan. beta(1-3)glucan is highly branched with beta(1-6) linkages constituting a
three-dimensional network with a large number of side-chains and ramifications. Other
polysaccharides such as chitin, galactomannan and beta(1-3/1-4)glucan are cross-linked to the
branched beta(1-3/1-6)glucan network. alpha(1-3)glucan and galactosaminogalactan are
composing the amorphous inter-fibrillar cement.
The current knowledge of the function of the enzymes involved in the biosynthesis of the A.
fumigatus cell wall and the role of the different polysaccharides in the cell wall organisation will
be discussed in my talk with special emphasis on the use of cell wall as a drug target or
immunostimulator.




                                                  35
S33
Biofilm matrix regulation by Candida albicans Zap1
Clarissa Nobile2, Jeniel Nett3, Aaron Hernday2, Oliver Homann2, Jean-Sebastien Deneault4, Andre
Nantel4, David Andes3, Alexander Johnson2 and Aaron Mitchell1
1 Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh PA 15213,
USA, Phone: 412-268-5844, FAX: 412-268-7129, e-mail: apm1@andrew.cmu.edu, Web:
http://www.cmu.edu/bio/faculty/mitchell.shtml
2 U of California San Francisco
3 University of Wisconsin
4 Biotechnology Research Institute

A biofilm is a surface-associated population of microorganisms embedded in a matrix of
extracellular polymeric substances. Biofilms are a major natural growth form of microorganisms
and the cause of pervasive device-associated infection. This report focuses on the biofilm matrix
of Candida albicans, the major fungal pathogen of humans.
We report here that the C. albicans zinc-response transcription factor Zap1 is a negative regulator
of a major matrix component, soluble beta-1,3 glucan, in both in vitro and in vivo biofilm models.
To understand the mechanistic relationship between Zap1 and matrix, we identified Zap1 target
genes through expression profiling and full genome chromatin immunoprecipitation.
Based on these results, we designed additional experiments showing that two glucoamylases,
Gca1 and Gca2, have positive roles in matrix production and may function through hydrolysis of
insoluble beta-1,3 glucan chains. We also show that a group of alcohol dehydrogenases Adh5,
Csh1, and Ifd6, have roles in matrix production: Adh5 acts positively and Csh1 and Ifd6,
negatively. We propose that these alcohol dehydrogenases generate quorum sensing aryl and acyl
alcohols that in turn govern multiple events in biofilm maturation. Our findings define a novel
regulatory circuit and its mechanism of control of a process central to infection.




                                                36
S34
Fungal vaccines : a critical view
Antonio Cassone
Infectious Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 299, Rome 00161, ITALY,
Phone: +390649387113, FAX: +390649387183, e-mail: cassone@iss.it, Web: www.iss.it

A number of vaccine formulations have been generated, which have been shown to be safe and
efficacious in pre-clinical models of fungal infections(1). Nonetheless, bringing a fungal vaccine
to clinical use in humans is not going to be an easy task, because of heavy constrains which go
from basic immuno-biological issues to priority strategies of vaccine manufacturers and connected
financial issues. The most basic obstacle stems from the fact that humans are already naturally
“vaccinated” (immunized) against the agents of fungal infections, due either to commensalism or
repeated natural exposures, and disease occurs because of the “loss” of previously acquired
immunity This makes an important difference with almost all other vaccines used in humans
which are designed to protect immunologically naïve subjects. The greatest medical need of a
fungal vaccine is not to immunize normal subjects but only those who have lost or at a great risk
of losing anti-fungal immunity. Thus, fungal vaccines should either be able to “repair” the
immunity lost in the immunocompromized subject or generate a novel immunity that can survive
even a deep immunodepression (“reparative immunization”). Coherently, the immunity induced
by the vaccination could, or even should, be different from the natural acquired one.
Following this line of thinking, we have generated a vaccine ( the Lam-CRM conjugate; 2) , using
beta,1-3-glucan , a vital polysaccharide possessed by all human pathogenic fungi. The vaccine has
been shown to induce in rodent models a protective immunity based on anti-beta,1-3-glucan IgG
which are absent or present in very low quantity in normal subjects(3). These antibodies are non-
opsonic , don’t favour the fungicidal activity of phagocytes and include isotypes with rather long
half-life in serum. They appear to exert protection by “neutralizing” some GPI cell wall proteins
exerting critical functions in cell wall integrity, hyphal formation and adherence to host tissues.
Overall, the Lam-CRM vaccine appears to confer protection by generating antibodies acting on
virulence properties of the fungus , and not relying ( at least not completely relying) on host
effector mechanisms. With these properties , this conjugate vaccine has the potential to become a
valuable tool to fight fungal infections in immunocompromized host.
1. Cassone, A. Lancet Infect. Dis. 2008 8:114-24.
2. Torosantucci, A. et al. J. Exp. Med. 2005 202:597-606.
3. Chiani,P. et al. Vaccine 2009 27:513-9.
4. Torosantucci, A. et al. PLoS One, 2009, revision stage.




                                                37
S35
Fungal genomics and natural product discovery: in search of novel antifungal agents
Terry Roemer
Infectious Disease, Merck & Co., Inc., 126 East Lincoln Ave., Rahway NJ 07065, USA, Phone: 1-
732-594-4906, FAX: 1-732-594-6708, e-mail: terry_roemer@merck.com

Natural products provide an unparalleled source of chemical scaffolds with diverse biological
activities and have profoundly impacted antimicrobial drug discovery. To further explore their
potential as a source for novel antifungal agents, we have surveyed natural products for antifungal
target-specific inhibitors using a chemical-genetics approach adapted to the fungal pathogen,
Candida albicans. Applying this C. albicans Fitness Test screening paradigm, natural product
fermentation extracts can be mechanistically-annotated to prioritize their potential progression for
chemical isolation and lead evaluation. As an example, the discovery and characterization of a
novel structural class of natural products, named parnafungins, will be discussed. Parnafungins
inhibit poly (A) polymerase as determined by biochemical and genetic means. Further,
parnafungins display potent and broad spectrum activity against diverse clinically-relevant fungal
pathogens and reduce fungal burden in a murine model of disseminated candidiasis. Thus,
mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling
brings a powerful strategy to natural product-based drug discovery. Genome Canada and Genome
Quebec are acknowledged for funding support.

References:
Jiang, B., et al. (2008) Chemistry and Biology 15, 363-74.
Parish, C., et al. (2008) J. Am. Chem. Soc. 130, 7060-66.
Adam, G., et al. (2008) J. Am. Chem. Soc.130, 16704-10.




                                                38
        SESSION 4

HOST-PATHOGEN INTERACTIONS




            39
S41
Mechanisms of the interaction of Aspergillus fumigatus with immune effector cells
Axel A. Brakhage
Molecular and Applied Microbiology, Leibniz-Institute (HKI), University of Jena,
Beutenbergstrasse 11a, Jena 07745, Germany, Phone: +49 (0)3641 532 1001, FAX: +49 (0)3641
532 0802, e-mail: axel.brakhage@hki-jena.de, Web: www.hki-jena.de

The improvement in transplant medicine and the therapy of hematological malignancies is often
complicated by the threat of infections caused by fungi. Species of the Aspergillus family account
for most of these infections and in particular Aspergillus fumigatus can be regarded as the primary
mould pathogen. Specific diagnostics are still limited as are the possibilities of therapeutic
intervention, leading to the fact that invasive aspergillosis (IA) is still associated with a high
mortality rate that ranges from 30 % to 90 %. Alveolar macrophages and neutrophilic
granulocytes are thought to be the major immune effector cells required for defence against IA.
However, until today it is still unknown by which mechanisms the immune effector cells kill A.
fumigatus. Recently, using genetic and cellular microbiological techniques we showed that it is
questionable that reactive oxygen intermediates (ROI) play a major role for neutrophils to kill A.
fumigatus. Therefore, our focus lies on the characterisation of phagosome lysosome fusion which
is reduced when wild-type conidia are phagocytosed by macrophages compared with distinct
mutant conidia. Also, we showed that A. fumigatus induces the production of nuclear extracellular
traps (NETs) and is able to evade the human complement system. In addition, a novel melanin
biosynthesis pathway was discovered which might contribute to virulence of A. fumigatus.




                                                40
S42
Phase-specific genes and intracellular survival strategies of Histoplasma capsulatum
William Goldman
Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill,
116 Manning Drive, Campus Box 7290, Chapel Hill NC 27599, USA, Phone: +1 919 966 9580,
FAX:        +1    919       962      8103,      e-mail:       goldman@med.unc.edu,   Web:
http://microimm.med.unc.edu/facultydetail.aspx?id=210

Histoplasma capsulatum is one of the best-studied dimorphic fungal pathogens, and most
biological work has focused on specific characteristics that enable the yeast form to be a
successful intracellular parasite of macrophages. New molecular genetic tools have allowed us to
evaluate the expression and prove the importance of two yeast phase-specific factors: CBP, a
secreted protein that is essential for virulence, and alpha-(1,3)-glucan, a virulence-associated cell
wall polysaccharide. However, the mechanisms that explain their role in fungal pathogenicity have
remained a complete mystery until recently.

We have now used NMR to solve the structure of CBP, revealing that this protein is a protease-
resistant homodimer and a member of the saposin family of lipid- and membrane-binding proteins.
It is likely that CBP is involved in lipid binding, lipid metabolism, and/or membrane remodeling
in the phagolysosomal compartment in which Histoplasma resides. We have taken two approaches
to study alpha-(1,3)-glucan: the first is a forward genetics strategy, using Agrobacterium-mediated
insertional mutagenesis, to identify genes implicated in the regulation, synthesis, and processing
of alpha-(1,3)-glucan. The second approach uses reverse genetics, combining fungal gene
disruption with mammalian RNA-interference, to study the genes involved in production of and
response to alpha-(1,3)-glucan. This work has revealed that alpha-(1,3)-glucan on the surface of
Histoplasma yeasts masks recognition of the underlying beta-glucan by dectin-1, a macrophage
pattern-recognition receptor that is critical in the innate immune response to fungi.




                                                 41
S43
Mechanisms of invasion of host cells by Candida albicans
Scott Filler1, Esteban Veiga2, Norma Solis1, Quynh Phan1, Jianing Sun3, Namrata Nayyar3, Emilia
Moreno-Ruiz4, Marta Galán-Díez2, Mira Edgerton5, Weidong Zhu1, Elena Fernandez-Ruiz2,
Pascale Cossart6 and Christophe d’Enfert4
1 Department of Medicine, Los Angeles Biomedical Research Institute, 1124 W. Carson St.,
Torrance CA 90502, USA, Phone: 01 310 222-6426, FAX: 01 310 782-2016, e-mail:
sfiller@ucla.edu
2 Department of Molecular Biology, Hospital Universitario de la Princesa, Madrid, Spain
3 School of Dental Medicine and Public Health and Health Professions, State University of New
York at Buffalo, USA
4 Institut Pasteur, Unité Biologie et Pathogénicité Fongiques, Paris, France
5 School of Dental Medicine and Public Health and Health Professions, State University of New
York at Buffalo
6 Institut Pasteur, Unité des Interactions Bactéries-Cellules, Paris, France

C. albicans invades endothelial and oral epithelial cells by inducing its own uptake. This
mechanism involves the binding of C. albicans Als3 and other proteins to N-cadherin on
endothelial cells and E-cadherin on epithelial cells. We have been investigating the role of host
cell clathrin in the uptake of C. albicans, and identifying fungal proteins other than Als3 that
induce this uptake. Using live-cell imaging and indirect immunofluorescence of host cells infected
with C. albicans, we observed that epithelial cell E-cadherin, clathrin, dynamin, and cortactin
accumulated at sites of fungal uptake. Similar proteins accumulated around C. albicans
internalized by endothelial cells, except that N-cadherin was recruited instead of E-cadherin.
Furthermore, clathrin, dynamin or cortactin depletion strongly inhibited C. albicans uptake by
epithelial cells. Finally, beads coated with Als3 were internalized in a clathrin-dependent manner.
Therefore, C. albicans hijacks the clathrin-dependent endocytic machinery to invade host cells.
The C. albicans HSP70 proteins, Ssa1 and Ssa2, are located on the surface of the organism where
they are targets of antimicrobial peptides. We investigated the roles of these proteins in C. albicans
host cell invasion and virulence. All mice infected intravenously with a ssa1/ssa1 mutant survived
after 21 days compared to a median survival of 7-8 days for mice infected with the wild-type
(WT), ssa2/ssa2, and ssa1/ssa1::SSA1 complemented strains. Mice infected with the ssa1/ssa1
mutant also had significantly reduced kidney, liver, and brain fungal burden. The ssa1/ssa1 mutant
had markedly impaired virulence in the mouse model of oropharyngeal candidiasis. Mice infected
orally with this strain had ~100-fold lower oral fungal burden after 5 days of infection compared
to mice infected with the control strains. The ssa1/ssa1 mutant had WT susceptibility to
environmental stressors and killing by HL-60 cells. However, it had significantly reduced capacity
to bind to cadherins, and to invade and damage endothelial and epithelial cells. Furthermore,
significantly more latex beads coated with recombinant Ssa1 were internalized by these cells
compare with beads coated with BSA. Therefore, Ssa1 functions as an invasin by binding to host
cell cadherins and mediating invasion of these cells. Ssa1 is also essential for normal C. albicans
virulence.




                                                 42
S44
Drosophila melanogaster as a model to study host-fungi interactions
Jessica Quintin, Samuel Liégeois, Ghulam Hussain, Sebastian Niehus, Marie Gottar, Vanessa
Gobert, Alexei Matskevitch and Dominique Ferrandon
UPR 9022 du CNRS IBMC, CNRS, 15, rue R. Descartes, Strasbourg F67084, France, Phone: 33 3
88 41 70 17, FAX: 33 3 88 41 70 17, e-mail: D.Ferrandon@ibmc.u-strasbg.fr

In flies, two NF-kappaB-like pathways, Toll and IMD, control the expression of potent
antimicrobial peptides as well as that of hundreds of genes during the systemic immune response
to a septic injury. The Toll pathway is required for the host defense against fungal and Gram (+)
bacterial infections. It provides protection against Candida and Cryptococcus infections.
The detection of infections is crucial to the initiation of the host immune response. Pattern
Recognition Receptors (PRRs) are thought to bind to relatively invariant microbial molecules such
as LPS, peptidoglycan, dsRNA, and lipoproteins. The activation of the Toll receptor by fungi is
mediated by GNBP3, a member of the Gram Negative Binding Protein/ß Glucan Recognition
Protein. GNBP3 mutants are sensitive to fungal and not to bacterial infections. This phenotype
correlates with a lack of induction of Drosomycin, a read-out of Toll pathway activation, in
response to a challenge with dead yeast. The overexpression of GNBP3 is sufficient to activate the
Toll pathway in the absence of infection. These data, together with the finding that recombinant
GNBP3 binds to long chains of ß(1,3) glucans, establish GNBP3 as a bona fide fungal PRR of the
Toll pathway. We have found that entomopathogenic fungi such as Beauveria bassiana are able to
bypass GNBP3 detection. A complementary pathway that senses the enzymatic activity of fungal
virulence factors and that leads to Toll pathway activation through the cleavage of the host
protease Persephone nevertheless detects them. Thus, Drosophila uses a dual sensor system to
detect fungal infections: GNBP3 senses the presence of microbial cell wall components whereas
Persephone detects the activity of fungal virulence factors. This strategy, which may result from
the evolution of host-pathogen interactions, is strikingly similar to that of plants and may be
widespread in the animal kingdom.
Drosophila can be used as a system for assessing the virulence of human fungal pathogens.
Examples of studies performed with Candida albicans and Candida glabrata will be presented,
using both systemic infection and a digestive tract colonization models.




                                               43
S45
Host defense against Candida albicans infections: from Drosophila to the patient
Mihai Netea
Radboud University Nijmegen Medical Center, Geert Grooteplein 8, Nijmegen 6500 HB,
Netherlands, Phone: +31 (0)24 3614652, FAX: +31 (0)24 3541734, e-mail: m.netea@aig.umcn.nl

Until a decade ago the innate host defense against pathogenic microorganisms was considered to
be a limited set of responses that aimed at containing an infection by primitive “ingest and kill”
mechanisms, giving time to the host defense to mount specific humoral and cellular immune
responses. It was not till the mid 90’s when the discovery of Toll-like receptors heralded a
revolution in our understanding of how microorganisms are recognized, and how the host defense
mechanisms are activated. Several major classes of pathogen-recognition receptors have now been
described, each with specific capabilities in recognizing conserved structures of bacteria. The
specific roles of the main families of receptors involved in antifungal host defense, theTLRs and
the C-type lectin receptors (CLRs), will be discussed. In addition, the role of polymorphisms and
deficiencies in these receptors for the susceptibility to Candida infections will be presented. These
complementary data will enable us to propose a model for understanding both the recognition
mechanisms of Candida albicans, as well as the pathways leading to the activation of host defense
during fungal infections.




                                                 44
           SESSION 5

SYSTEMS BIOLOGY IN PATHOGENESIS




              45
S51
Systems genetics of yeast filamentation
Timothy Galitski
Institute for Systems Biology, 1441 N 34th Street, Seattle WA 98103, USA, Phone: 1 206 732
1206, FAX: 1 206 732 1260, e-mail: tgalitski@systemsbiology.org

We have developed and tested a computational method that integrates molecular interactions,
which provide paths for information flow, and genetic interactions, which reveal active
information flows and reflect their functional consequences. These complementary data types are
integrated to model the transcription network controlling filamentous-form cell differentiation in
yeast. Genetic interactions were inferred from linear decomposition of gene expression data and
were used to direct the construction of a molecular interaction network mediating these genetic
effects. This network included both known and novel regulatory influences, and the model
successfully predicted genomic expression and filamentation phenotypes of novel combinations of
mutations.




                                               46
S52
Deciphering cellular networks and pathways using yeast functional genomics
Brenda Andrews, Timothy Hughes and Charles Boone
Centre for Cellular & Biomolecular Research, University of Toronto, Rm 230 160 College Street,
Toronto ON M5S 3E1, CANADA, Phone: 416-978-8562, FAX: 416-946-8253, e-mail:
brenda.andrews@utoronto.ca, Web: http://www.utoronto.ca/andrewslab/

Determining how combinations of genetic variants or perturbations manifest themselves,
particularly in the context of human disease, is a formidable challenge. To define general
principles of genetic networks, our group has focused on the systematic identification of genetic
interactions in the budding yeast. Synthetic genetic array (SGA) analysis provides a high
throughput approach for systematic analysis of genetic interactions in budding yeast. We have
used SGA analysis to generate a large-scale view of synthetic lethal interactions in yeast, assessing
both essential and non-essential genes. We estimate that a global synthetic lethal genetic network
will contain on the order of 200 000 genetic interactions, and we are testing this prediction by
using SGA to complete the synthetic lethal map. We have also expanded our SGA platform to
encompass other types of genetic interactions and to include cell biological phenotypes and
quantitative read-outs of the activity of specific biological pathways. In one project, we combined
SGA with a high-content screening (HCS) platform, to monitor morphological phenotypes of the
growing mitotic spindle in both single gene deletion mutants and in selected double mutant arrays,
sensitized for spindle defects. HCS enables virtually any pathway that can be monitored with a
fluorescent reporter to be assessed quantitatively within the context of numerous genetic and
environmental perturbations. In principle, screens could be devised to specifically monitor
pathways relevant to fungal pathogenesis. In a more general sense, genetic interaction maps will
provide a global view of the cell, enabling a systematic understanding of fungal biology.




                                                 47
S53
Functional genomics of fluconazole sensitivity in Candida glabrata using a library of
transcription factor knock-outs.
Biao Ma, Andrew McDonagh, Emily Cook, Melanie Puttnam and Ken Haynes
Microbiology, Imperial College London, The Flowers Building, London SW7 2AZ, United
Kingdom, Phone: +44 (0)20 7594 2072, FAX: +44 (0)20 7594 3095, e-mail:
k.haynes@imperial.ac.uk

Systems biology seeks to understand how the components of a system interact to facilitate the
function of that system. In order for this to happen many tools (biological and modelling) must be
in place. In Saccharomyces cerevisiae systems biology has been facilitated by the existence of a
large number of tools eg micro-arrays, protein arrays, knock-out collections, ORF’s on demand,
tagged libraries, an extremely well supported database (yeastgenome.org), a huge literature, and
not least an extremely collegial community. As yet most of these resources are not yet available
for a pathogenic fungus, although significant efforts have been made for Candida albicans.

We have focused our efforts on Candida glabrata, an increasingly common pathogen. Here we
report on the development of a medium-throughput method for the construction of bar-coded
knock-out strains in C. glabrata. This method was applied to the construction of a library of 186
transcription factor knock-outs, that was subsequently used to functionally screen for fluconazole
sensitivity. A novel automated microtitre plate based growth assay was used. Strains were
incubated overnight to stationary phase (18 h, 37ºC, 180 rpm, in YPD) and then sub-cultured (18
h, 37ºC, 180 rpm) in deep-well microtitre plates containing YPD +/- fluconazole (5 - 100ug /ml).
Optical density measurements were recorded bihourly on an automated liquid handling platform,
and data were fitted to a three parameter (slope, midpoint and upper asymptote) logistic function
of growth using non-linear regression. Strains showing significant, fluconazole specific growth
defects were identified in null strains either by a) a complete lack of growth in the presence of
fluconazole, but not YPD alone or b) significantly higher midpoint (p <0.05) estimates than wild-
type strains in fluconazole but not YPD. A total of 12 mutants had fluconazole specific growth
defects, indicating that the deleted transcription factor plays some role in mediating fluconazole
resistance. Validation of the assay was provided by the observation, that as predicted from work in
C. albicans and S. cerevisiae respectively pdr1 and gal11 nulls were more sensitive than wild-type
cells. Intriguingly C. glabrata aro80 mutants also display increased sensitivity to fluconazole,
possibly implicating aromatic amino acid catabolism in antifungal drug resistance.




                                                48
S54
Molecular networks of T-helper cell differentiation: from experiments to computational
models and back
Thomas Höfer1, Dorothea Busse1, Edda Schulz1, Luca Mariani1, Max Löhning2, Alex Scheffold2
and Andreas Radbruch2
1 Modeling of Biological Systems, German Cancer Research Center, Im Neuenheimer Feld 280,
Heidelberg 69120, Germany, Phone: 004962215451380, FAX: 004962215451487, e-mail:
t.hoefer@dkfz-heidelberg.de, Web: www.dkfz.de
2 German Rheumatology Research Center, Berlin

To mount appropriate adaptive immune responses, T helper lymphocytes differentiate into various
lineages of effector and memory cells, including Th1, Th2 and Th17 cells. These cellular decisions
are governed by complex and dynamic molecular networks. We have employed iterative cycles of
experimental work and mathematical modelling to dissect the functioning of such networks. I will
discuss studies on (1) the differentiation of Th1 cells, and (2) the IL-2 cytokine network
established between Th cells and regulatory T cells. Our results show how the interplay between
computer simulations and experimental testing of mechanistic hypotheses helps discover novel
molecular interactions and identifies systems-level regulatory switches. Feedback loops between
gene regulation and cell signalling emerge as crucial regulatory motifs.




                                               49
        ABSTRACTS

POSTERS AND WORKSHOP TALKS




            50
P1A
Hybridization and phylogenetics in predicting pathogen emergence on a novel host
Amanda Gibson1, Tatiana Giraud1 and Michael Hood2
1 Ecologie, Systématique et Evolution, UMR 8079 CNRS, ESE, Université de Paris-Sud XI,
Bâtiment 360, Orsay 91405, France, Phone: +33 (0) 6 7157 2710, FAX: +33 (0) 1 6915 4697, e-
mail: Amanda.Gibson@u-psud.fr
2 Department of Biology, McGuire Life Sciences Building, Amherst College, Rts 9 & 116,
Amherst, MA 01002-5000, USA, Phone: 1-(413) 542- 8538, e-mail: mhood@amherst.edu

Emerging infectious diseases frequently trace their origins to a cross-species transmission event
between the native and novel hosts of a pathogen. Yet the processes that allow the pathogen to be
sustained on a novel host remain obscure, complicated by numerous physiological and ecological
factors that limit opportunities for pathogen adaptation to novel hosts. Previous studies have
proposed several traits that may enhance the potential of pathogens to bridge the gap between two
host species; most notably, they point to an expanded host range displayed by hybrid pathogens
and to a close phylogenetic relationship of the pathogen’s native and novel hosts. In this study, we
examined successful infection of novel hosts of the plant genus Silene following artificial
inoculation with multiple species of the fungal pathogen Microbotryum violaceum. Molecular
identification of pathogens revealed that the majority of infections on novel hosts resulted from
hybrid combinations of Microbotryum species. The pathogens’ phylogenetic relationships also
revealed that the genetic distance of the two haploid members of a hybrid pathogen was
significantly negatively correlated with the hybrid’s frequency of infection. Furthermore, the
genetic distance between the pathogen’s native and novel hosts was found to be significantly
negatively correlated with the frequency of infection. As a whole, this study demonstrates the
importance of the evolutionary divergence of both the host and pathogen lineages in the
emergence of new diseases and the significance of hybridization in facilitating cross-species
transmission.




                                                51
P2B
G+C content variation in the Saccharomycotina
Denise B. Lynch1, Mary E. Logue1, Geraldine Butler1 and Kenneth H. Wolfe2
1 School of Biomolecular and Biomedical Science, University College Dublin, Donneybrook,
Dublin 04, Ireland, Phone: +353 1 716 6838, FAX: +353 1 716 6701, e-mail: denise.lynch@ucd.ie
2 Smurfit Institute of Genetics, University of Dublin, Trinity College, Dublin 2, Ireland. e-mail:
khwolfe@tcd.ie

Local similarities of G+C (guanine and cytosine) content of DNA has been shown to correlate
with gene density and recombination frequencies. A number of models have been suggested in an
attempt to explain the origin of these blocks or isochores, but as of yet, there is no clear
explanation. The bakers’ yeast, Saccharomyces cerevisiae, has previously been shown to contain
significant regional variation of G+C content throughout its genome, particularly on chromosome
III [Sharp et al., 1993]. The significance of G+C variations needs to be studied in more detail.
Comparisons to other genomes will allow us to identify conservation of these variations, which
may in turn help us to understand the functional significance. Here, we look at the silent site G+C
content (GC3s) variation of the coding regions of four closely related Saccharomycotina; S.
cerevisiae, S. bayanus, S. mikatae and S. paradoxus, and compare the patterns to those observed in
9 Candida species. We use a sliding window of 15 adjacent genes that are orthologues of S.
cerevisiae and plot the variations along each of the 16 chromosomes. We show that these species
display similar patterns of variations along their chromosomes, but with S. bayanus showing
consistently and significantly higher GC3s percentages. This appears to correspond with the
phylogenetic distances, as S. bayanus is the most distantly related of these four species. C.
albicans and C. dubliniensis have a surprisingly similar distribution of isochors, but slight
differences may be related to their differential roles in infection of mammalian hosts. We have
identified potential centromere signals in C. lusitaniae and Pichia stipitis. It has been difficult to
date to identify centromeres in Candida species using purely sequence-based approaches. We
propose to further investigate these regions using additional comparative analysis.

Sharp, P. M., Lloyd, A. T. (1993) Nucleic Acids Research 21(2): 179-183




                                                 52
P3C
Rapid quantification of viable Candida spp. cells in whole blood by immunomagnetic
separation combined with solid-phase cytometry
Lies Vanhee1, Katrien Lagrou2, Wouter Meersseman3, Hans Nelis1 and Tom Coenye1
1 Ghent University, Laboratory of Pharmaceutical Microbiology, Harelbekestraat 72, Ghent 9000,
Belgium, Phone: +32 9 264 8142, FAX: +32 9 264 8195, e-mail: Lies.Vanhee@UGent.be
2 Department of Medical Diagnostic Sciences, University Hospitals Leuven, Herestraat 49, B-
3000 Leuven, Belgium
3 Medical Intensive Care Unit, University Hospitals Leuven, Herestraat 49, B-3000 Leuven,
Belgium

Candida spp. are now the fourth most common source of nosocomial bloodstream infections in
critically ill patients. Therefore, rapid isolation and identification of this pathogenic yeast are
crucial. Traditional diagnostic procedures based on blood cultures lack speed and a sufficiently
low detection limit to ensure reliable and early diagnosis of invasive Candida infections. A two
hour method based on immunomagnetic separation (IMS) and solid-phase cytometry (SPC) has
been developed. In a first step, Candida cells present in a whole blood sample (max. 15 ml) are
magnetically labelled with a primary anti-Candida FITC conjugated antibody and a secondary
anti-FITC Microbead conjugated antibody. Subsequently, Candida cells are separated from their
matrix using the MACS technology. The obtained suspension is filtered and the retained cells are
stained with the dye ChemChrome V6, which allows for the labelling of all viable cells. Finally,
the membrane filter is scanned by a solid-phase cytometer and each detected cells is
microscopically inspected. To verify the sensitivity of this approach, blood samples spiked with
different amounts of Candida albicans, C. glabrata, C. krusei, C. parapsilosis, or C. tropicalis
were analysed. These tests confirmed that the detection limit for all Candida spp. was as low as 1
cell/ml of blood. Additionally, applying the assay to blood samples spiked with other fungi
including Aspergillus, Cryptococcus and Fusarium spp. proved its specificity. To demonstrate the
diagnostic value of this method, blood samples from patients with candidemia will be analysed
using a traditional blood culture method and the two hour IMS-SPC protocol. In conclusion, we
developed a rapid and highly sensitive method for the diagnosis of candidemia. The procedure has
been validated on spiked blood samples and analysis of patient samples is ongoing.




                                                53
P4A
Genes implicated in RNA interference in Cryptococcus neoformans
Frédérique Moyrand and Guilhem Janbon
Unité des Aspergillus, Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France, Phone: 33
(0)145688356, FAX: 33 (0)145688420, e-mail: janbon@pasteur.fr

Cryptococcus neoformans is a basidiomycete pathogenic yeast and its genome has been sequenced
recently. Comparative analysis of the genome sequence identified homologous sequences for each
of the major RNA associated protein complexes. This analysis suggested that the RNA
metabolism in C. neoformans has the same complexity as compared to higher eukaryotes. The
functionality of the RNA silencing pathway has been previously demonstrated (1). Here, we
looked for genes potentially implicated in the RNA silencing pathway in C. neoformans. We
identified 2 putative Argonaute encoding genes, 2 Dicer homologues and one putative RNA
dependent RNA polymerase encoding gene. We constructed different corresponding mutant
strains and demonstrated that some of these genes are necessary for RNA silencing in C.
neoformans whereas some are dispensable. However none of these genes seems to regulate any of
the classical virulence factors of this yeast.

1. Liu, H., T.R. Cottrell, L.M. Pierini, W.E. Goldman, and T.L. Doering. 2002. RNA interference
in the pathogenic fungus Cryptococcus neoformans. Genetics 160:463-470.




                                              54
P5B
Analysis of the role of Upc2 in the hypoxic response in Candida albicans
John Synnott, Alessandro Guida, Siobhán Mulhern-Haughey and Geraldine Butler
UCD School of Biomolecular and Biomedical Science, Conway Institute, University College
Dublin, Belfield, Dublin D4, Ireland, Phone: +353 (0)1 716 6838, FAX: +353 (0)1 716 6701, e-
mail: john.synnott@ucd.ie, Web: http://www.ucd.ie/biochem/gb/Lab/

The pathogenic yeast Candida albicans produces filaments in response to hypoxic (low oxygen)
conditions. In S. cerevisiae, the levels of haem and of sterols are used to sense oxygen. We
screened a set of C. albicans strains carrying knockouts of transcription factors (provided by A.
Mitchell and D. Sanglard), and some additional candidate genes, for defects in response to
hypoxia. Disrupting BCR1 results in hyperfilamentation in hypoxia, whereas deleting UPC2
completely abolishes filamentation. Upc2 is required for expression of several genes in the
ergosterol pathway, whose expression is induced by hypoxia (Silver et al and Setiadi et al).
We confirmed that hypoxic conditions induce expression of cell wall metabolism, ergosterol
synthesis, and glycolytic genes. We then compared the transcriptional profile of upc2 knockout
and wild type cells grown in hypoxic conditions, and we showed that the three classes of genes
respond differently. Hypoxic induction of the ergosterol pathway requires UPC2. Expression of
three members of the CFEM family of cell wall genes (RBT5, PGA7 and PGA10) is also induced
by hypoxia, and induction requires both UPC2 and BCR1. Hypoxic induction of glycolytic genes
does not appear to require UPC2 or BCR1. We next used ketaconazole to lower sterol levels, and
compared the transcriptional response to the hypoxic profile. Ketaconazole treatment mimics the
hypoxic response of the ergosterol pathway, but not the glycolytic pathway. Expression of
glycolytic genes is induced by hypoxia but is repressed by ketoconazole. The decrease in
glycolytic gene expression in not affected by deleting UPC2. Our results suggest that the hypoxic
response of the ergosterol pathway and of cell wall genes is at least partly signalled by lowering
sterol levels. The hypoxic response of glycolytic genes however is regulated in a different manner.




                                                55
P6C
Evidence that the Vancouver Island Cryptococcus gattii outbreak has expanded into the
United States Pacific NW
Edmond J. Byrnes III1, Wenjun Li1, Yonathan Lewit1, Sheryl A. Frank1, Robert J. Bildfell2, Beth
A. Valentine2, Sarah West3, Thomas G. Mitchell1, Kieren A. Marr4 and Joseph Heitman1
1 Molecular Genetics and Microbiology, Duke University, 312 CARL Building, Research Drive
DUMC, Durham NC 27713, United States, Phone: 919-768-3981, FAX: 919-684-5458, e-mail:
edmond.byrnes@duke.edu
2 Oregon State University, Corvallis, Oregon
3 Oregon Health and Science University, Portland, Oregon
4 Johns Hopkins University, Baltimore, MD

Cryptococcus neoformans and Cryptococcus gattii are common fungal mammalian pathogens. C.
neoformans is more prevalent, associated with pigeons in nature and a frequent cause of
meningitis in immunocompromised patients, whereas C. gattii is geographically restricted to
tropical and subtropical regions, associated with trees and usually infects immunocompetent
individuals. Since 1999, an outbreak of C. gattii on Vancouver Island, British Columbia, has
become endemic, caused numerous human and veterinary infections, and spread to mainland
British Columbia. The outbreak isolates of C. gattii were characterized as molecular type
VGIIa/major or VGIIb/minor. Beginning in 2006, human and veterinary cases have emerged in
Washington State and Oregon. Using high-resolution multilocus sequence typing at a minimum of
eight unlinked loci, we determined that most of these strains were VGIIa/major or VGIIb/minor,
which provides direct evidence for the emergent spread of C. gattii from Vancouver Island to the
Pacific Northwest of the United States. In addition, five isolates unique to Oregon and related to
the VGIIa/major genotype form a novel cluster, which we have termed VGIIc. In addition, highly
variable non-coding regions are under examination to further detect genomic differences among
the VGIIa/major outbreak isolates. Continued analysis of veterinary, human, and environmental
isolates from the region is ongoing, and the C. gattii Working Group of the Pacific Northwest has
been established as a multidisciplinary effort to study the emergence. This unusual outbreak in a
temperate climate raises concern about further expansion in the region and illustrates how
microbial pathogens emerge in novel geographic locales.




                                               56
P7A
Large scale synthetic genetic analysis of the RAM signaling network in C. albicans
Yeissa Chabrier-Roselló1, Nike Bharucha2, Anuj Kumar2 and Damian Krysan1
1 Pediatrics and Microbiology/Immunology, University of Rochester School of Medicine &
Dentistry, 601 Elmwood Ave, Rochester NY 14642, United States, Phone: 939-579-4608, FAX:
585-273-1104, e-mail: yeissa_chabrierrosello@urmc.rochester.edu
2 Life Sciences Institute, University of Michigan, Ann Arbor, MI

Candida albicans is the most common opportunistic fungal pathogen, and an important cause for
morbidity and mortality among the immunocompromised patient population. In recent years
genetic manipulation of C. albicans has greatly advanced, but now a challenge has become to
understand the complex genetic interactions that carry out and regulate cellular processes. Here we
describe a novel strategy for large scale synthetic genetic analysis in C. albicans based on
complex haploinsufficiency (CHI). CHI occurs when a strain containing heterozygous mutations
at two separate loci displays a more severe phenotype than strains containing single heterozygous
mutations at the same loci. In this study we utilize this method to better understand the
mechanisms responsible for yeast-to-hypha transition, which is tightly associated with the onset of
disease in this pathogen.
A variety of regulatory pathways are involved in this characteristic transition, but here we focused
on the RAM network (Regulation of Ace2p and Morphogenesis). The RAM network primarily
regulates the transcription factor Ace2p, but neither the transcriptional targets nor the inter-
pathway connections of this pathway have been clearly defined in C. albicans. We have applied
our novel strategy to a large-scale transposon mutagenesis-based CHI screen of C. albicans strain
heterozygous at CBK1, the RAM protein kinase, to identify double mutants with decreased hypha
formation. To date, we have screened 6528 independent transformants, identified 441 clones with
decreased hyphae formation relative to cbk1/CBK1, and sequenced 39 unique CBK1-interacting
gene candidates.
Among the set of candidate CBK1-interacting genes, 15 potential targets (12 of which are novel)
of the Cbk1p-dependent transcription factor Ace2p have been identified, strongly supporting the
ability of this approach to identify genes that have a direct mechanistic relationship to the RAM
network. We have also found that genes regulated by the cAMP-PKA pathway are over-
represented (9/39) in our initial set of CBK1-interactors, suggesting that the RAM and cAMP-
PKA pathways may carry out inter-related functions during hyphal development. These data
provide new insights into the role of the RAM pathway in hyphal development and more
importantly, provide a new tool for the genetic analysis of this important human pathogen.




                                                57
P8B
The human pathogenic fungus Aspergillus fumigatus produces pyomelanin via the tyrosine
degradation pathway
Jeannette Schmaler-Ripcke, Venelina Sugareva, Sophia Keller, Juliane Macheleidt, Thorsten
Heinekamp and Axel A. Brakhage
Molecular and Applied Microbiology, Hans Knoell Institute, Beutenbergstr. 11a, Jena 07745,
Germany, Phone: +49 (0)3641 532 1095, FAX: +49 (0)3641 532 0803, e-mail:
thorsten.heinekamp@hki-jena.de, Web: http://www.hki-jena.de

Aspergillus fumigatus is the most important air-borne fungal pathogen of immunosuppressed
humans. This mould possesses specific physiological and molecular characteristics including the
biosynthesis of a certain type of melanin, i.e., dihydroxynaphthalene (DHN) melanin. This
pigment biosynthesis pathway contributes in a complex manner to the pathogenicity of A.
fumigatus. Melanins are pigments of high molecular weight that are formed by oxidative
polymerization of phenolic and/or indolic compounds. They protect the fungus against different
stresses, e.g. oxidants, extreme temperature, and antifungal agents.
Here, we show that A. fumigatus is able to produce pyomelanin, a second type of melanin, via the
tyrosine degradation pathway. Pyomelanins are synthesized from L-tyrosine or L-phenylalanine
through p-hydroxyphenylpyruvate and homogentisic acid. Analysis of the genomic organization
of the genes involved in tyrosine degradation revealed that the genes are arranged in a cluster.
Transcription of these genes is strongly induced by tyrosine. To investigate the pyomelanin
biosynthesis pathway in detail, we deleted the genes encoding essential enzymes for pigment
production, homogentisate dioxygenase (hmgA) and 4-hydroxyphenylpyruvate dioxygenase
(hppD). In the hmgA deletion strain, HmgA activity was completely abolished and the
accumulation of homogentisic acid drastically increased pigment formation. By contrast,
homogentisic acid and pyomelanin were not observed in an hppD deletion mutant. Germlings of
the hppD deletion mutant showed increased sensitivity against reactive oxygen intermediates.
However, the hmgA deletion mutant characterized by enhanced pyomelanin formation, did not
show reduced sensitivity against ROI. Therefore, the role of pyomelanin in scavening of ROI in
vivo and its putative role in virulence remains to be elucidated.




                                              58
P9C
Regulation of PMT genes encoding protein-O-mannosyltransferases in Candida albicans
Pilar D. Cantero1,2 and Joachim F. Ernst1
Institut fuer Mikrobiologie, Heinrich Heine Universitaet, Universitaetsstrasse 1, Duesseldorf
40225, Germany, Phone: +49 211 8114835, FAX: +49 211 8115176, e-mail: soile@usal.es
1Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
2Departamento de Microbiología y Genética, Universidad de Salamanca, Salamanca, Spain.

Protein mannosyltransferases (Pmt proteins) catalyze the addition of the first molecule of mannose
to Ser and/or Thr residues of O-mannosylated proteins.
Five Pmt isoforms have been described in Candida albicans and classified into three subfamilies
(Pmt1-, Pmt2- and Pmt4-subfamilies). Pmt isoforms largely do not carry out redundant functions
but have specific roles in O-mannosylation of secretory proteins involved in growth,
morphogenesis, resistance to cell wall disturbing agents and antifungals, as well as in virulence.
To investigate transcriptional regulation of PMT genes during alteration or damage of the cell
wall, PMT transcript levels were determined by qPCR in different cell wall mutants or in a wild-
type strain treated with cell wall inhibitors. While the PMT1 transcript was upregulated in all
mutants and conditions, the PMT4 transcript was downregulated. The PMT2 transcript was
downregulated especially if N-glycosylation was altered. To further investigate this regulation, the
5´-transcriptional start site of all PMT genes was determined and their promoter regions were
cloned upstream of the RLUC reporter gene. Analysis of reporter activity under different
growth/stress conditions revealed that PMT transcript levels are regulated by cell wall alterations
on the level of transcriptional initiation. Data will be presented on deletion analyses to delineate
PMT promoter regions important for transcriptional regulation.




                                                59
P10A
Hypoxic adaptation by Efg1 regulates biofilm formation of Candida albicans
Catrin Stichternoth and Joachim F. Ernst
Institut fuer Mikrobiologie, Heinrich Heine Universitaet, Universitaetsstr. 1, Duesseldorf 40225,
Germany, Phone: +49 211 8114835, Fax: +49 211 8115176, e-mail: C.Stichternoth@uni-
duesseldorf.de
Catrin Stichternoth and Joachim F. Ernst
Institut für Mikrobiologie, Molekulare Mykologie, Heinrich-Heine-Universität, D-40225
Düsseldorf, Germany

Hypoxia is encountered frequently by Candida albicans during systemic infection of the human
host. We tested if hypoxia allows biofilm formation by C. albicans, which is a major cause for the
perseverance and antifungal resistance of its infections. Using an in vitro biofilm system we
unexpectedly discovered that several positive regulators of biofilm formation during normoxia
including Tec1, Ace2, Czf1, Och1 and Als3 had no or little influence for biofilm development
during hypoxia, irrespective of carbon dioxide levels, indicating that C. albicans biofilm
pathways differ depending on oxygen levels. In contrast, the Efg1 and Flo8 regulators were
required for both normoxic and hypoxic biofilm formation.
To explore the role of Efg1 during hypoxic and/or biofilm growth we determined transcriptomal
kinetics following release of EFG1 expression by a tet-on system. During hypoxia, Efg1 rapidly
induced all major classes of genes known to be associated with normoxic biofilm formation,
including genes involved in glycolysis, sulfur metabolism and antioxidative/peroxisomal
activities, as well as genes for iron uptake. The results suggest that hypoxic adaptation mediated
by the Efg1/Flo8 regulators is required even during normoxic biofilm development, while hypoxic
biofilm formation in deep tissues or in organs may generate foci of C. albicans infections.




                                               60
P11B
Transcriptional profiling in C. parapsilosis
Claudia Jürgensen, Alessandro Guida and Geraldine Butler
School of Biomolecular and Biomedical Science, University College Dublin, Conway Institute,
Belfield Dublin 4, Ireland, Phone: +353 1 716 6838, FAX: +353 1 283 7211, e-mail:
claudia.jurgensen@ucd.ie, Web: www.ucd.ie

The genome sequence of Candida parapsilosis (haploid genome size 13 Mb with 7 chromosomes)
was           recently         determined         by          the         Sanger          Institute
(http://www.sanger.ac.uk/sequencing/Candida/parapsilosis/). The C. parapsilosis genome is
among the most complete eukaryotic genome sequences available. The first sequence release in
2005 included 24 contigs of over 2 kb in length and was used to make preliminary annotations of
the C. parapsilosis genome. More recently, the sequence has been assembled into 8 supercontigs
larger than 200kb, with one supercontig corresponding to each chromosome.
Currently, there are two preliminary genome annotations based on the first sequence release, one
of which originated in our lab (identified by the tag “cpar”) and one from the Broad Institute
(identified by the tag “cpag”). We used these annotations to design gene-specific oligonucleotides
for microarrays manufactured by Agilent. Each gene is represented by two oligonucleotides and
each oligonucleotide is spotted on the array twice, generating four expression values for every
gene. The arrays were successfully used for several whole genome expression analyses in C.
parapsilosis, such as determining the transcriptional profile in hypoxic conditions, during
ketoconazole treatment and during growth in low iron conditions, as well as identifying targets of
the transcription factor Bcr1.
We are now using next-generation sequencing (Illumina) to help re-annotate the C. parapsilosis
genome, and to further our understanding of gene expression. PolyA-RNA has been isolated from
C. parapsilosis cells grown in rich media (YPD). We are currently using this to generate double
stranded cDNA, which will be fragmented and subjected to high-throughput Illumina sequencing.
The resulting short reads will then be mapped onto the genome sequence, using software tools
such as SSAHA (Sequence Search and Alignment by Hashing Algorithm) and MAQ (Mapping
and Assembly with Quality). This RNA-Seq approach will help us to identify all the transcribed
regions in the C. parapsilosis genome, at least during growth on rich media. This will greatly
increase the accuracy of the genome annotation, as well as provide a new method for comparing
transcriptional profiles.




                                                61
P12C
Role of C. albicans cyclin CLB4 in S-phase initiation
Ayala Ofir and Daniel Kornitzer
Molecular Microbiology, Technion Faculty of Medicine, 2, Efron St., Haifa 31096, Israel, Phone:
+972 (0)4 829 5258, FAX: +972 (0)4 829 5254, e-mail: danielk@tx.technion.ac.il

Cyclin-dependent kinases (CDKs) are key regulators of eukaryotic cell-cycle progression. The
cyclin subunit activates the CDK and also imparts to it, at least in some cases, substrate
specificity. S. cerevisiae, an organism where the role of individual cyclins is fairly well
understood, contains nine cyclins (three G1 cyclins and six B-type cyclins) capable of activating
the main cell-cycle CDK, Cdc28. The C. albicans genome, in contrast, contains a Cdc28
homologue, three homologues of the S. cerevisiae G1 cyclins, but only two B-type cyclin
homologues, CaClb2 and -4. In addition, Sol1, a CDK inhibitor (CDKI) analogous to the main S.
cerevisiae CDKI Sic1, was recently identified. Sic1 inhibits both the mitotic cyclin ScClb2 and the
S-phase cyclin ScClb5. We find that Sol1, like Sic1, inhibits activity of both CaClb2- and CaClb4-
Cdc28. Since the S-phase cyclin ScClb5 has no obvious sequence homolog in C. albicans, we set
up a series of functional assays to determine which C. albicans cyclin carries out the function of
ScClb5 in S-phase initiation. We first tested the ability of all the cyclin-CDK complexes to
phosphorylate the ScClb5-specific substrate Cdc6. We found that both CaClb2 and -4 are active
towards this substrate, but that CaClb4 seems to be more active, relative to the generic CDK
substrate Histone H1. Next, we tested two C. albicans proteins that are homologous to known
ScClb5 substrates (CaCdc6 and CaMcm4). Similar to ScCdc6, both proteins were significantly
better substrates of CaClb4 than CaClb2 (~ 20 fold higher relative phosphorylation signal), and
were not phosphorylated at all by the G1 cyclin-CDK complexes. As an additional test of CaClb4
vs. CaClb2 specificity for S-phase initiation, we substituted the CLB5 coding region in S.
cerevisiae with either CaCLB2 or CaCLB4, and compared these strains to the clb5^ knockout.
Only CaCLB4 was able to suppress the S-phase delay of the clb5^ mutant. Intriguingly, the S.
cerevisiae clb5^::CaCLB4 cell population, which was initially haploid, rapidly accumulated
diploid cells, which took over the population within a few tens of generations. Thus, in spite of
limited sequence homology, we identified CaClb4 as the functional Clb5 homolog of C. albicans.
Whether the diploidization of S. cerevisiae in the presence of CaClb4 reflects a novel function of
this cyclin, or is due to mis-regulation of the C. albicans cyclin in a heterologous environment,
remains to be investigated.




                                                62
P13A
Role of introns in the regulation of gene expression in Cryptococcus neoformans
Estelle Mogensen, Frédérique Moyrand and Guilhem Janbon
Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, FRANCE, Phone: +33 1 45 68
83 56, FAX: +33 1 45 68 84 20, e-mail: estelle.mogensen@pasteur.fr, Web: http://www.pasteur.fr/

Cryptococcus neoformans is an opportunistic pathogen found in the environment. This
basidiomycete synthesizes a polysaccharide capsule which has been shown to be a major virulence
attribute. The gene CAS3 is required for the O-acetylation of glucuronoxylomannan, the main
component of the capsule. In the course of our experiments, we showed that the expression of the
CAS3 cDNA was not able to complement a cas3 mutation suggesting that introns play a major
role in gene expression in C. neoformans. Indeed, introns represent key elements for the regulation
of gene expression in eukaryotes. Genomic analyses of C. neoformans revealed that 98% of the
genes contain introns, with an average of 5 introns per gene (Loftus et al., 2005). Moreover, C.
neoformans possesses components of mRNA processing machineries (such as exon junction
complex, RNA interference and nonsense-mediated decay effectors) that are homologues of those
present in more complex eukaryotic organisms. Therefore, C. neoformans is a good yeast model to
study the role of introns in modulating gene expression.
In order to establish a model of CAS3 expression, we constructed a library of mutants harbouring
a copy of CAS3 harbouring a variable number of introns. We have shown that CAS3 expression
not only requires the presence of introns but is dependent on the number and the location of the
introns. Moreover, some introns regulate positively CAS3 expression whereas others function as
negative regulators. We have shown by run-on experiments that transcription level of CAS3 is
similar in all the intron mutants tested and the wild-type strain. We are now investigating the role
of the exon junction complex and the nonsense-mediated decay in the regulation of CAS3
expression.

Loftus, B., et al., (2005), Science, 307, 1321




                                                 63
P14B
Longitudinal Study of Candida Density in HIV-infected Patients receiving highly active
antiretroviral therapy (HAART)
Nadja Melo1, Hideaki Taguchi2, Oslei Almeida3, Rogerio Pedro4 and Jacks Jorge3
1 Immunology, Institute of Life Science, Swansea University, Singleton Park, Swansea SA2 8PP,
UK, Phone: +44 01792 368578, FAX: +44 01792 301022, e-mail: nadjarm@yahoo.com
2 Research Center for Fungi Pathogenic and Microbial Toxicoses – Chiba University - Japan
3 Dental Faculty UNICAMP – Piracicaba – SP- Brazil
4 Medical Sciences Faculty, UNICAMP, Campinas Brazil

The present study investigated the correlation between the levels of Candida (CFU/mL) and
Candida spp. prevalence in saliva obtained from HIV-infected patients receiving highly active
antiretroviral therapy (HAART). In addition Candida counts were correlated with clinical
manifestations associated with HIV infection and parameters such as salivary flow, smoking,
immunological biomarkers, sulfamethoxazole and trimethoprim prophylactic usage and HIV
counts. One hundred and eighty eight patients were enrolled, 93 of them were followed up for 17
months. Candidosis was found in 32% of the patients and the CFU/ml count was significantly
higher (Wilcoxon test, p=0.001) in these patients compared with counterparts with no oral
manifestation of disease. Candida levels showed a significant correlation with white cells counts
(p=0.004), Oxalacetic Glutamic Transaminase dosage (p=0.01), Pyruvic Glutamic Transaminase
dosage (p=0.006), and CD+4 count (p=0.03). Oral candidosis also showed significant correlation
with the CFU/mL density in the longitudinal analysis, and Candida levels were reduced over time.
Possibly this reduction was due to long term under HAART and oral health advices including
usage of oral chlorexidine could contribute to it. Candida albicans was obtained from 78.5% of
the patients investigated, while 24.2% were colonized with non-C. albicans spp.. Some members
of the patient cohort yielded multiple Candida spp.. Another important finding was the CFU/mL
density in the multiple C. albicans and non-C. albicans colonization group yielded higher counts
when compared to colonization with C. albicans only. Some non-albicans strains showed low
susceptibility to antifungal agents such as C. tropicalis and C. krusei, with MIC80 range of 4-
64ug/mL and 2-64ug/mL, respectively. C. glabrata CFU/ml counts for instance, were particularly
high (1811.7 +/- 3392.2 CFU/m) and this yeast exhibited notoriously high MIC80 range to
fluconazole (4-32ug/mL). CFU/mL quantification parameters showed correlation with some
systemic and local parameters indicating therefore that oral and systemic conditions should be
followed up since the initial phases of HIV infection.




                                               64
P15C
Dual-function protein: a putative translation elongation factor with glutathione s-transferase
activity protects Aspergillus fumigatus against oxidative stress
Grainne O' Keeffe, Christoph Jöchl, Kevin Kavanagh and Sean Doyle
Biology and National Institute for Cellular Biotechnology, National University of Ireland
Maynooth, Maynooth, Co. Kildare 00000, Ireland, Phone: +353 (0)1 708 3140, FAX: +353 (0)1
708 3845, e-mail: grainneokeeffe@gmail.com

Aspergillus fumigatus is an opportunistic pathogen predominantly affecting immunocompromised
individuals. Sequencing of the genome has led to an increased understanding of the organism;
however the functions of many genes remain unknown. A putative translation elongation factor
1Bgamma (EF1Bgamma, termed elfA; 750 bp) has been found to be expressed, and to exhibit
glutathione-s-transferase (GST) activity, in A. fumigatus (Carberry, S, et al. (2006), Biochem
Biophys Res Commun, 341, 1096). Normally, EF1Bgamma plays a key role in the elongation step
of protein synthesis. Our hypothesis is that elfA may also play a role in regulating the cellular
redox state adjacent to the ribosome during protein synthesis. Consequently, elfA was disrupted in
the A. fumigatus ATCC46645 using a bipartite construct containing overlapping fragments of a
pyrithiamine resistance gene (ptrA). Southern Blot analysis, through the use of a digoxigenin
labelled probe, was used to confirm the generation of an elfA mutant (deltaelfA) at the genome
level. This probe was specific for a PstI-digested fragment of 4376 bp in the wild-type and 1969
bp in deltaelfA, respectively. The availability of the mutant has facilitated phenotypic analysis of
elfA function. A. fumigatus wild-type and deltaelfA were grown on AMM plates with the oxidants
H2O2 (1 mM- 5 mM) and menadione (20 microM- 50 microM). After 72 hr incubation at 37°C,
deltaelfA was significantly more sensitive to H2O2 (p=0.0016) and menadione (p=0.0032) than
the wild-type strain. These results implicate elfA in the oxidative stress response in A. fumigatus.
Further phenotypic analysis is currently underway, as are complementation studies, to further
explore the function for elfA.
In receipt of an Embark PhD Scholarship from IRCSET.




                                                65
P16A
A systematic approach to identify virulence and drug resistance genes in the human fungal
pathogen Candida glabrata
Tobias Schwarzmüller1, Ingrid Frohner1, Helmut Jungwirth2, Walter Glaser1, Toni Gabaldon3 and
Karl Kuchler1
1 Christian Doppler Laboratory, Max F. Perutz Laboratories, Medical University of Vienna, Dr.
Bohr-Gasse 9/2, Vienna 1030, AUSTRIA, Phone: 00431-4277-61818, FAX: 00431-4277-9618, e-
mail:                      Tobias.Schwarzmueller@meduniwien.ac.at,                      Web:
http://www.meduniwien.ac.at/medbch/MolGen/kuchler/
2 University of Graz, Zentrum für Molekulare Biowissenschaften, Graz, AUSTRIA
3 Centro de Investigación Príncipe Felipe CIPF, Valencia, SPAIN

Candida glabrata (C.g) is an opportunistic human fungal pathogen. Like Candida albicans, it can
cause life-threatening systemic infections in immunocompromised individuals and is inherently
resistant to antifungal therapy. However, the molecular basis of C.g virulence and antifungal
resistance is not well understood. Based on the C.g genome sequence, we have initiated a large-
scale reverse genetic approach to identify novel virulence genes in this non-filamenting pathogen.
Based on bioinformatic analysis, we have deleted C.g genes having functional orthologues in the
related yeast Saccharomyces cerevisiae, in particular cell wall genes, signalling cascades and
transcriptional regulators. This way, we have generated a bar-coded C.g deletion strain collection
currently comprising more than 400 strains. Deletion strains were analyzed for growth phenotypes
on a variety of media containing different compounds, for morphology and drug sensitivity. We
identified C.g genes implicated in metal ion or detergent tolerance and resistance to cell wall-
perturbing compounds or antifungals. Moreover, phenotypes related to a host-pathogen interaction
situation were analyzed using an in vitro assay detecting reactive oxygen species (ROS) after
incubation with primary mouse macrophages. Interestingly, this ROS assay identified C.g mutants
defective in counteracting the accumulation of host-derived ROS produced by macrophages upon
C.g interaction. Hence, the corresponding genes may modulate fungal virulence through their
possible role in defense against host-derived ROS. Finally, the virulence characteristics of all these
strains will be further analyzed by in vivo mice experiments in the near future.

This work is supported by Christian Doppler Research Society and the ERA-Net Pathogenomics
project FunPath through the Austrian Science Foundation (FWF-I125-B09).




                                                 66
P17B
Monitoring amino acid and SPS-sensor controlled virulence of Candida albicans using insect
model host systems
Francisco J. Alvarez, Monica Davis, Kicki Ryman, Ylva Engström and Per O. Ljungdahl
Cell Biology, Wenner-Gren Institute, Svante Arrheniusväg 16-18, Stockholm 106 91, Sweden,
Phone: +46 8 16 28 36, FAX: +46 8 15 98 37, e-mail: javier.alvarez@wgi.su.se, Web:
http://www.wgi.su.se/

The Candida albicans plasma membrane-localized SPS-sensor responds to extracellular amino
acids and induces the endoproteolytic processing of the two latent cytoplasmic transcription
factors Stp1 and Stp2. Processing removes negative regulatory motifs present in the N-terminal
domains of these factors enabling them to target the nucleus where they modulate gene expression
by binding upstream activating sequences in the promoters of SPS-sensor regulated genes.
Processed Stp1 activates the expression of genes required for the catabolic utilization of
extracellular proteins, including secreted aspartyl proteases (SAPs) and oligopeptide transporters.
Processed Stp2 activates the expression of amino acid permease genes encoding proteins that
catalyze transport of amino acids into cells. In contrast to Stp2, Stp1 levels vary inversely with
amino acid availability, a consequence of STP1 expression being under nitrogen control. These
findings indicate that cells use the SPS-sensor to differentially control two discrete metabolic
pathways for the assimilation of nitrogen, and that cells preferentially use extracellular amino
acids when they are available. To test the role of the SPS sensing pathway during virulent
infections we have used Drosophila melanogaster and Galleria mellonella as mini-host models.
Systemic (injection of fungal strains into adult Drosophila flies and Galleria larvae) and oral
infection (feeding Drosophila flies on fungal lawns) strategies have been established and
optimized. Insects infected with wildtype Candida exhibit reduced viability; enhanced lethality is
not observed when insects are similarly challenged with Saccharomyces cerevisiae or heat-killed
Candida preparations. In comparison to wildtype Candida strains, isogenic mutant strains lacking
various SPS sensing pathway components exhibit significantly attenuated virulence. These insect
models represent simple and robust experimental systems to examine host-pathogen interactions.




                                                67
P18C
Sexual reproduction and recombination in the opportunistic fungal pathogen Aspergillus
fumigatus
Céline M. O'Gorman1, Hubert T. Fuller1 and Paul S. Dyer2
1 UCD School of Biology & Environmental Science, University College Dublin, Science Centre
(West), Belfield, Dublin 4, Ireland, Phone: +353 (0)1 716 2350, FAX: +353 (0)1 716 1153, e-
mail: celine.ogorman@ucd.ie
2 School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD, UK

Aspergillus fumigatus is a saprotrophic fungus whose spores are ubiquitous in the atmosphere. It is
also an opportunistic human pathogen in immunocompromised individuals, causing potentially
lethal invasive infections. The species is only known to reproduce by asexual means, but there has
been accumulating evidence for recombination and gene flow from population genetic studies,
genome analysis, the presence of mating-type genes and expression of sex-related genes in the
fungus. We have discovered that A. fumigatus possesses a fully functional sexual reproductive
cycle that leads to the production of cleistothecia and ascospores [1]. The teleomorph (sexual
stage) has been assigned to the genus Neosartorya on the basis of phylogenetic relatedness and the
morphology of the cleistothecia and ascospores and named Neosartorya fumigata. The species has
a heterothallic breeding system; isolates of complementary mating type are required for sex to
occur. The defined conditions for sexual reproduction are growth on Parafilm-sealed Oatmeal agar
plates with incubation in darkness at 30 °C for 6 months. Increased genotypic variation resulting
from recombination was demonstrated in N. fumigata ascospore progeny from an Irish
environmental subpopulation. The ability of A. fumigatus to engage in sexual reproduction is
highly significant in understanding the biology and evolution of the species. The presence of a
sexual cycle provides an invaluable tool for classical genetic analyses and will facilitate research
into the genetic basis of pathogenicity and fungicide resistance, with the aim of improving
methods for the control of aspergillosis. The results also yield insights into the potential for sexual
reproduction in other ‘asexual’ fungi, many of which are of great economic and medical
importance. The isolation of fresh cultures combined with concerted laboratory crossing efforts of
compatible mating types may lead to a sexual revolution for many of these supposedly ‘asexual’
fungi.

[1] O’Gorman, C., et al., (2009), Nature, 457, 471




                                                  68
P19A
Open platform technologies for unbiased analyses of gene expression in fungal pathogens
Elena Lindemann1, Christian Grumaz1, Bettina Rohde2, Steffen Rupp1, Johannes Regenbogen3
and Kai Sohn1
1 MBT, Fraunhofer IGB, Nobelstr. 12, Stuttgart 70569, Germany, Phone: +49 (0)711 970 4055,
FAX: +49 (0)711 970 4200, e-mail: kso@igb.fhg.de
2 GATC-Biotech, Jakob-Stadler-Platz 7, 78467 Konstanz, Germany
3 Baxter AG, Wagramerstr. 17-19, 1220 Vienna, Austria

The DNA-microarray technology has emerged as the application of choice for the identification of
differentially expressed genes. However, the generation of microarrays requires the availability of
completely sequenced and annotated genomes thus reducing the choice of organisms to be studied
and restricting the analysis only to annotated transcriptional units. Here, we describe a
Multidimensional Electrophoretic System of Separation for the Analysis of Gene Expression
(MESSAGE) as well as a Parallel Sequencing System for the Analysis of Gene Expression
(PASSAGE). Both systems represent two open platform technologies for global transcriptional
profiling that do not necessarily depend on annotated genome sequence information, hence
making this system universally applicable for any eukaryotic organism. MESSAGE is based on
the two-dimensional electrophoretic separation of complex cDNA samples according to molecular
weight using non-denaturing polyacrylamide gel electrophoresis in the first dimension, and to GC
content, by use of denaturing gradient gel electrophoresis (DGGE) in the second dimension.
Subsequent quantitative analysis of spot patterns derived from different samples allows for the
identification of differentially expressed transcripts as well as de-novo mapping of yet not annoted
transcriptional units. Quantitative data on differential transcription highly correlate with
corresponding DNA-microarray or qRT-PCR data. Applying MESSAGE for transcriptional
profiling of fungal morphogenesis we could identify yet uncharacterized transcriptional units as
well as differentially expressed open reading frames during blastospore-to-hyphae transition in C.
dubliniensis for which the annotation of the genome sequence is still in progress. Complementary,
sensitive global transcriptional profiling using next-generation parallel sequencing (PASSAGE)
revealed differential digital expression profiles derived from 10 ng of total RNA in C. albicans
and C. dubliniensis. Taken together, MESSAGE as well as PASSAGE not only allows for
unbiased transcriptional profiling, but also is predestined for cross-species comparative gene
expression analyses in all kind of pathogenic fungi.




                                                69
P20B
New tools for studying the Candida albicans genetic code
Ana Rita Bezerra, João Simões and Manuel Santos
CESAM - Department of Biology, University of Aveiro, Campus Universitário de Santiago,
Aveiro 3810-193, Portugal, Phone: +351 234 370 350 (lab 22754), FAX: +351 234 372 587, e-
mail: armbezerra@ua.pt

Candida albicans and other Candida species changed the identity of the leucine CUG codon to
serine through an ambiguous codon decoding mechanism. In this case, a unique tRNA
(tRNACAGSer) decodes leucine CUG codons as serine. Interestingly, the tRNACAGSer is
recognized by both leucyl- and seryl-tRNA synthetases and it is aminoacylated in vivo with both
serine (97%) and leucine (3%). We have demonstrated that such tRNA ambiguity is incorporated
into proteins and that the C. albicans proteome has a statistical nature. It is not yet clear whether
proteome variation is relevant for C. albicans pathogenesis, however the potential of CUG
ambiguity to alter cell wall proteins and remodel surface antigens may help C. albicans evading
the immune system. In order to clarify this important question we are determining the maximum
amount of ambiguity that can be tolerated by C. albicans. For this, we engineered tRNAs that
decode CUG codons as leucine and developed a new reporter system to monitor CUG ambiguity
in vivo. This reporter system is based on gain of function of Green Fluorescent Protein (GFP) and
allows one to quantify CUG ambiguity using fluorescence microscopy and flow cytometry.
Preliminary results show that this reporter is quantitative and its easy manipulation allows one to
monitor CUG ambiguity under different physiological conditions, in different C. albicans strains
and also in mice during C. albicans infection. The data from these studies will be presented at the
meeting.

Acknowledgements: ARB is supported by the Portuguese Foundation for Science and Technology
through the PhD grant REF: SFRH/BD/39030/2007.




                                                 70
P21C
The mating-type locus in the Candida parapsilosis species group
Sixiang Sai, Conor McGee and Geraldine Butler
School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin,
Belfield, Dublin DUBLIN4, Ireland, Phone: +353879454870, FAX: +353-1-2837211, e-mail:
sixiang.sai@ucd.ie, Web: http://www.ucd.ie/biochem/gb/Lab/

Candida species are an important cause of nosocomial infection and rank fourth in bloodstream
infections in the United States. C. parapsilosis strains have historically been categorized as group
I, II, or III on the basis of molecular fingerprinting. Group I strains which include the type strain
CLIB214 are predominant in clinical isolates. Analysis of levels of heterozygosity and of
mitochondrial genome architecture supports the hypothesis that the three groups represent three
different species, and recently group II and group III isolates have been renamed as the new
species C. orthopsilosis and C. metapsilosis. All three species are distantly related to C. albicans.
C. parapsilosis isolates appear to contain only one mating-type like locus (MTL) idiomorph
(MTLa), and the MTLa1 region is a pseudogene. We have investigated the organization of the
MTL in 16 isolates of C. orthopsilosis and 18 isolates of C. metapsilosis. Approximately 10 kb
surrounding the MTL of each isolate was amplified by long PCR. Molecular fingerprinting of C.
orthopsilosis isolates show that they fall into two groups (Group A and Group B), which may
represent individual species. This is currently being investigated further. The C. orthopsilosis
isolates were further catergorised as either MTLa, MTLalpha and MTLa/alpha. MTLa idiomorphs
from both Group A and Group B have been sequenced, and they are intact (including the a1 gene).
The sequence of the C. orthopsilois MTLalpha idiomorph is currently being completed. The C.
metapsilosis isolates are all heterozygous at MTL. The MTLalpha idiomorph has been sequenced
and is intact. We are currently attempting to clone and sequence the MTLa idiomorph. Our results
suggest that the loss of MTLa1 and the entire MTLalpha idiomorph is restricted to C. parapsilosis.
The lack of sequence heterozygosity in this species may be related to its virulence.




                                                 71
P22A
Epidemiology and antifungal susceptibility profile of Candida parapsilosis, C. orthopsilosis
and C. metapsilosis, in a tertiary care hospital
Ana Silva, Isabel Miranda, Carmen Lisboa, Cidália Pina-Vaz and Acácio Rodrigues
Department of Microbiology, Medicine Faculty, University of Porto, Al. Prof. Hernâni Monteiro,
Porto 4200-319, PORTUGAL, Phone: 00351917123337, FAX: 00351225513662, e-mail:
anatpsilva@hotmail.com, Web: www.fmup.pt

C. parapsilosis, which has emerged as an important agent of nosocomial infection, formed a
complex of three genetically distinct groups (I, II and III). Recently, C. parapsilosis groups were
renamed as distinct species: C. parapsilosis, C. orthopsilosis and C. metapsilosis. In our country,
no data pertaining to the distribution and antifungal susceptibility of such species is yet available.
In the present study we describe the incidence and distribution of C. parapsilosis, C. orthopsilosis
and C. metapsilosis within a set of 175 clinical and environmental isolates from different care
units of Hospital S. João, previously identified by conventional methods as C. parapsilosis. We
also evaluated the susceptibility profile to fluconazole, voriconazole, posaconazole, amphotericin
B and the echinocandins caspofungin and anidulafungin. Of the 175 isolates, 160 (91.4%) were
identified as C. parapsilosis, 4 (2.3%) as C. orthopsilosis and 5 (2.9%) as C. metapsilosis. Six
isolates corresponded to species other than C. parapsilosis group. Interestingly, all isolates from
blood cultures corresponded to C. parapsilosis. Regarding the antifungal susceptibility profile, 9
(5.6%) C. parapsilosis strains were susceptible-dose dependent or resistant to fluconazole and
only a single strain displayed a multi-azole-resistant phenotype. Two (1.3%) C. parapsilosis
strains were amphotericin B resistant. All C. orthopsilosis and C. metapsilosis isolates were
susceptible to azoles and amphotericin B. Concerning echinocandins, the levels of no
susceptibility of C. parapsilosis, C. orthopsilosis and C. metapsilosis were high. We demonstrated
the low incidence of C. orthopsilosis and C. metapsilosis in clinical isolates, especially in blood
cultures. The described differences in antifungal susceptibility were not relevant, thus suggesting
that the routine discrimination within C. parapsilosis complex is not mandatory for the clinical
laboratory.




                                                 72
P23B
Crystal structures of the SerRS explain proteome tolerance to a genetic code alteration in
Candida albicans
Manuel A. S. Santos
CESAM - Department of Biology, University of Aveiro, Campus Universitário Santiago, Aveiro
3810-193, Portugal, Phone: +351234370771, FAX: +351 234 372 587, e-mail: msantos@ua.pt,
Web: http://www.ua.pt/ii/rnomics/

Several species of the genus Candida changed the identity of the leucine CUG codon to serine.
This unique sense-to-sense eukaryotic genetic code alteration reassigned approximately 16,000
CUG codons in the ancestor of Candida species. Remarkably, in Candida albicans the CUG
codon remains ambiguous and under normal growth conditions incorporation of leucine and serine
ranges between 3-5% and 95-97%, respectively. Engineered C. albicans cells tolerate up to 28%
of leucine incorporation at CUG positions, which represents an increase of 28,000 folds in codon
decoding error; without visible decrease in growth rate. These observations raised the intriguing
question of how the Candida ancestor tolerated reassignment of CUG codons from leucine to
serine and how extant C. albicans tolerate such high levels of CUG ambiguity. In other words,
why are Candida proteins tolerant to CUG misreading? In order to answer these important
questions, we have determined crystal structures of the C. albicans SerRS, whose gene contains a
CUG codon, with serine and leucine at CUG position and carried out comparative stability assays
of wild type and mutant enzymes. The crystal structures showed that the residue encoded by the
CUG codon was partially buried in a position where both serine and leucine could be
accommodated without significant disruption of the structure and stability of the enzyme. These
results were further supported by functional complementation of Saccharomyces cerevisiae SerRS
gene (SES1) knockout by the C. albicans CaSES1 gene. Also, molecular modelling and
comparative genomics studies of various C. albicans proteins, whose genes encode one or more
CUG codons, showed that serine and leucine incorporation at CUG positions does not cause
disruption of protein structure. Finally, statistical modelling of the impact of CUG ambiguity on
the C. albicans poteome showed that the genome distribution of CUGs minimizes the negative
effects of misreading. These data are in sharp contrast with the negative effects of CUG ambiguity
in S. cerevisiae proteins where serine insertion at leucine CUG positions causes major proteome
disruption and is lethal at low level (3% misincorporation). Therefore, the high tolerance of C.
albicans proteins to CUG ambiguity results from a unique genome distribution of CUG codons
that permits accommodation of both serine and leucine at CUG positions.




                                               73
P24C
Fungal phylogenomics
Marina Marcet-Houben and Toni Gabaldón
Bioinformatics and Genomics department, CRG (Center for Genomic Regulation), Doctor
Aiguader, 88, Barcelona 08003, Spain, Phone: +34 93 316 02 82, FAX: +34 93 316 00 99, e-mail:
mmarcet@crg.es, Web: http://www.crg.es/comparative_genomics

In the last years many fungal genomes have been sequenced providing a great amount of new
information. Currently, the complete genomes of more than 60 fungal species have been made
available and more are on its way, paving the way for gaining a broader understanding on this
important kingdom. Phylogenetic tools can be applied on this large quantity of data in order to
help us address many evolutionary and biological questions. Here we present results from several
phylogenomic analyses using 60 complete fungal genomes. Firstly, we constructed a species tree
using 69 wide-spread protein families by applying an alignment concatenation method and
performing Maximum Likelihood analyses on the composite alignment. Results were similar to
that of other, previously published phylogenetic analyses. Secondly, we built several fungal
phylomes (i.e a complete collection of phylogenies for each gene in a genome). For this we
applied an automated pipleine used before in the construction of the human phylome. Briefly, for
each gene of the genome, homologs are searched in a database formed by proteomes of the species
we wish to include. Subsequently, we align the sequences and clean the less conserved places of
the alignment. This trimmed alignment is then used to build phylogenetic trees using a maximum
likelihood approach. Phylomes can have many uses. For instance they can be used in order to
predict accurate, phylogeny based orthology relationships. We do that applying a tree scanning
algorithm. The resulting predictions can be used in different studies like the annotation of newly
sequenced genomes. Phylomes can also give an overview of which nodes in the species tree are
more robust. This analysis is often not congruent with bootstrap values and offers us additional
information on the reliable topology for the species tree. Finally we predicted the orthology
relationships between yeasts and different fungi and compared them to the golden standard given
by YGOB. All the data related to the fungal phylomes can be found in our database, phylomeDB
(www.phylomedb.org), which allows a user-friendly access to trees, alignments and orthology
predictions for all the phylomes we have constructed. Currently, five Saccharomyces cerevisiae
phylomes with different taxonomic scopes are available. A Candida glabrata phylome has been
built for the FunPath consortium and will be accessible in the future. Some other phylomes for
other clinically-relevant fungi are on its way.




                                               74
P25A
Tracing the path of centromere evolution
Kaustuv Sanyal1, Sreedevi Padmanabhan1, Jitendra Thakur1 and Rahul Siddarthan2
1 Molecular Biology & Genetics, JNCASR, Jakkur, Bangalore 560064, INDIA, Phone: +91 80
2208 2878, FAX: +91 80 2208 2766, e-mail: sanyal@jncasr.ac.in, Web:
http://www.jncasr.ac.in/sanyal
2 Institute of Matematical Sciences, Chennai 600113, INDIA

The centromere (CEN), that serves as the chromosomal attachment site of spindle microtubules,
plays a crucial role in chromosome segregation during mitosis and meiosis. Understanding
centromere structure/function after its first molecular characterization almost three decades ago is
still far from complete. Several lines of evidence suggest that centromere formation cannot be
solely governed by the DNA sequence, rather many other genetic and epigenetic factors are
involved. To better understand the mechanisms involved in centromere identity, its maintenance
and propagation, we have identified and analyzed centromeres of several Candida species. Our
studies suggest that centromere structures of two of these species, C. albicans and C. dubliniensis,
are different from those of other organisms. We have recently shown that in spite of having a very
high degree of similarity in DNA sequence in these two closely related yeasts, the centromere
sequences diverged more rapidly than any other regions in the genome. We propose that this rapid
evolution of centromeres, which work in highly species-specific manner, may serve as a driving
force for speciation. More recently, we have identified centromeres of another closely related
organism, C. tropicalis. Preliminary results suggest that centromere structure of this species
provides a missing link to a simple “point” centromere of S. cerevisiae and a more complex
regional centromere of fission yeast S. pombe.




                                                75
P26B
Dissociable subcomplex Rpb4/7 of RNA polymerase II affects morphogenesis in Candida
albicans
Manimala Sen, Vijender Singh and Parag Sadhale
Microbiology and Cell Biology, Indian Institute of Science, C V Raman Road, Bangalore KA
560012, INDIA, Phone: +91 80 22932292, FAX: +91 80 23602697, e-mail:
parag.sadhale@gmail.com

Candida albicans is pathogenic yeast that shows several morphological forms under a variety of
different conditions. The hyphal morphology is found to be associated with the pathogenicity of
the organism. Homologs of a large number of genes affecting the pseudohyphal morphogenesis of
the model organism Saccharomyces cerevisiae also have been shown to affect the morphogenesis
in C. albicans. We have been studying the role of RNA pol II subunits in regulating gene
expression involved in pseudohyphal morphogenesis. Two subunits Rpb4 and Rpb7, make up the
subcomplex which is a conserved component of eukaryotic RNA polymerase II. These two
subunits have been characterized in detail in the model system S. cerevisiae. The link between
levels of these subunits and the pseudohyphal transition in S. cerevisiae has been reported by our
group. Although there is significant conservation between the homologs of the two proteins at the
sequence level there is substantial deviation in their functionality in the two yeasts. Genetic
complementation, over-expression and genome wide expression studies reveal these differences.
We discuss the potential role of the homologs of the Pol II subunits in C. albicans.




                                               76
P27C
Detection of Pneumocystis jirovecii by flow cytometry
Joana Barbosa1, Joana Barbosa2, Claudia Bragada3, Ana Teresa Silva1, Sofia Costa - Oliveira1,
Acacio Gonçalves Rodrigues1, Acacio Gonçalves Rodrigues4, Cidalia Pina - Vaz1 and Cidalia
Pina - Vaz3
1 Department of Microbiology, Faculty of Medicine, University, Alameda Prof. Hernani
Monteiro, Porto 4250, Portugal, Phone: +351225513600, FAX: +351225513601, e-mail:
gui75@sapo.pt
2 Escola Superior de Saúde Jean Piaget, Vila Nova Gaia
3 Department of Microbiology, Hospital S. João, Porto
4 Burn Unit, Department of Plastic and Reconstructive Surgery, Hospital S. João, Porto

BACKGROUND: Pneumocystis jirovecii is responsible for severe pneumonia in
immunocompromised patients. Acquisition of P. jirovecii organisms may occur via airborne route
or inhalation; its diagnosis is based on epifluorescence microscopy visualization of specific
stained organisms in clinical specimens. Such methods are time-consuming, cumbersome and
subject to human error, especially when samples yield a low number of organisms.
OBJECTIVES: Optimization of a flow cytometric protocol for the detection of P. iirovecii in
respiratory samples.
METHODS: 420 respiratory samples were subjected to immunofluorescence staining (IFS) and
flow cytometry analysis (FC). Clinical samples were firstly mixed with mucolytic agent (N –
acetyl – L – cystein, Merck®) before analysis. For IFS, smears were stained with Detect IFTM kit
Pneumocystis carinii (Axis – Shield Diagnostics Limited, United Kingdom) according to the
manufacturer’s instructions. For FC, samples were filtered (Partec CellTrics®), stained with serial
concentrations of the same fluorochrome and analysed in a FACSCalibur cytometer (Becton
Dickinson, Canada) at FL1 (525 nm – green fluorescence). After mixing bacteria (Escherichia coli
and Staphylococcus aureus) and fungi (Candida albicans) cross-reactions were investigated.
Suspensions were also stained with 20 &#956;g/ml of propidium iodide (PI, Sigma) (a marker of
death) and analysed at FL3 (670 nm-red). Clinical outcome was evaluated.
RESULTS: The optimal specific-antibody concentration for FC analysis was 10 µg/ml and no
cross-reactions occurred with bacteria or fungi. Using the two fluorescent probes simultaneously,
dead organisms showed double fluorescence (green and red). FC showed higher sensitivity than
IFS since we found 88 positive samples against 80. Such discrepant results were in patients that
have favourable outcome after receiving specific therapy.
CONCLUSIONS: A new approach is now available to detect P. jirovecii from respiratory
samples, allowing the simultaneous assessment of its infective status.




                                                77
P28A
Identification and characterization of genes controlling genome stability in the human
fungal pathogen Candida albicans
Mélanie Legrand, Arnaud Firon and Christophe d'Enfert
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: mlegrand@pasteur.fr

Candida albicans is the single most important human fungal pathogen. In clinical settings, a 25-
60% mortality rate associated with disseminated infection has been reported, due in part to
increasing resistance to the most popularly used drug, fluconazole. Genome changes such as
aneuploidy, translocations, loss of heterozygosity, or point mutations are often observed in
commensal isolates as well as clinical isolates that have become resistant to antifungal drugs.
Because genome plasticity is likely to be crucial for the pathogenicity of this obligate diploid
fungus, an insight in genome maintenance is necessary.
We have developed a new overexpression-based approach to characterize the molecular
mechanisms allowing/leading to genome instability in C. albicans. Our aim is to identify genes
whose overexpression leads to an increase in Loss-Of-Heterozygosity events. To this end, 204
genes have been selected based on their GO annotations, relevant to mitotic and meiotic
recombination, DNA replication and DNA repair. The ORFs were cloned into a C. albicans
integrative plasmid, using the GATEWAY cloning technology, which facilitates the high-
throughput cloning of PCR products by recombination. This plasmid carries a tetracycline-
regulatable promoter, allowing doxycycline-induced overexpression of the cloned ORFs, as well
as a unique 20bp DNA barcode. In addition to conducting standard screening assays to analyse
mutants individually, the barcodes enable us to investigate the fitness of the overexpression clones
from a single pooled culture under various growth conditions.
The resulting barcoded overexpression plasmids have been introduced in a C. albicans strain
engineered for monitoring Loss-Of-Heterozygosity events at the ADE2 locus. The strain carries
only one allele of the ADE2 gene, allowing us to correlate the appearance of red sectors with an
increase in genome instability upon overexpression of a specific gene. Moreover, alterations of a
variety of phenotypes related to genome integrity such as cell cycle, DNA repair or telomere
maintenance are also being tested.
Here, we will report the results of the screens we are conducting on the overexpression collection.
Identification of genes whose overexpression triggers genome alterations will allow the
characterization of new players in genome maintenance and therefore a better understanding of
how genomic instability may contribute to C. albicans success as a commensal and pathogen.




                                                78
P29B
SidL, an acetyltransferase involved in biosynthesis of the intracellular siderophore
ferricrocin in Aspergillus fumgatus
Michael Blatzer, Markus Schrettl and Hubertus Haas
Department for Molecularbiology, Biozentrum Innsbruck Medical University, Fritz Pregl Strasse
3, Innsbruck A 6020, Austria, Phone: +43 (0)512 9003 70231, FAX: +43 (0)512 9003 73100, e-
mail: Michael.Blatzer@i-med.ac.at
Divison of Molecular Biology/Biocenter, Medical University of Innsbruck, Fritz-Pregl-
Str. 3, A–6020 Innsbruck/Austria, Phone: ++43-512-9003-70205, Fax: ++43-512-
9003-73100, Email: michael.blatzer@i-med.ac.at

Virtually all organisms require iron as indispensable cofactor for various metabolic processes. The
opportunistic fungal pathogen Aspergillus fumigatus produces two major siderophores (low
molecular-mass ferric iron chelators): it excretes triacetylfusarinine C for iron uptake and
accumulates ferricrocin for intracellular iron storage. Biosynthesis of both triacetylfusarinine C
and ferricrocin has previously been shown to be crucial for virulence of A. fumigatus.
Here, we report the functional characterization of a new component of the fungal siderophore
biosynthetic machinery Afu1g04450, termed SidL. SidL is conserved in siderophore-producing
but not non-siderophore producing ascomycetes. The C-terminal half of SidL shows similarity to
acetylases involved in bacterial siderophore biosynthesis, e.g. Escherichia coli IucB (a
hydroxylysine acetylase required for aerobactin biosynthesis) and PvdY (a hydroxyornithine
acetylase required for pyoverdin biosynthesis), and the hydroxyornithine:anhydromevalonyl
coenzyme A-transacylase SidF that is essential for triacetylfusarine C biosynthesis. Deletion of
sidL in A. fumigatus reduced ferricrocin biosynthesis during iron starvation and blocked
ferricirocin biosynthesis during iron-replete growth. Furthermore, sidL-deficiency blocked
conidial ferricrocin accumulation under strict iron-replete conditions but not when mycelia were
transferred from iron-depleted to iron- replete conditions before sporulation. In contrast, SidL-
deficiency had no effect on triacetylfusarinine C production. The expression of sidL was affected
neither by iron availability nor the iron regulator SreA.
Taken together, these data show that SidL is a constitutively expressed hydroxyornithine acetylase
involved in ferricrocin biosynthesis. Moreover, the data indicate the existence of a second
hydroxyornithine acetylase, the activity of which is induced by iron starvation. This study
identified a novel component of the fungal siderophore biosynthetic machinery and revealed
unexpected complexity.
This work was supported by Austrian Science Foundation Grant FWF-P18606-B11.




                                                79
P30C
Fungal genomes at PhylomeDB
Marina Marcet-Houben, Jaime Huerta-Cepas, Salvador Capella-Gutierrez and Toni Gabaldón
Bioinformatics and Genomics, Centre for Genomic Regulation (CRG), Dr. Aiguader, 88,
Barcelona 08003, SPAIN, Phone: +34 933160281, FAX: +34 93 396 99 83, e-mail:
tgabaldon@crg.es, Web: www.crg.es/comparative_genomics

Phylogenetic studies provide very valuable information about the evolutionary relationships
between homologous genes of different species. Among other applications, phylogenies can be
exploited to map duplication and speciation events and thus infer orthology relationships, to
determine the evolutionary relationships among taxa and even to reconstruct ancestral sequences.
Therefore the generation of complete collections of phylogenetic trees for all genes encoded in a
single genome (phylome) is a useful resource for many researchers. PhylomeDB is a database of
complete phylomes derived for different genomes within a specific taxonomic range. All
phylomes in the database are built using a high-quality phylogenetic pipeline that includes
evolutionary model testing and alignment trimming phases. For each genome, PhylomeDB
provides the alignments, phylogentic trees and tree-based orthology predictions for every single
encoded protein. Currently PhylomeDB offers several fungal genomes included those of
Saccharomyces cerevisiae and Candida glabrata, which have been generated using information
from 60 fully-sequenced fungal genomes. Other fungal genomes will be soon incorporated. S.
cerevisiae PhylomeDB entries can be accessed through PhylomeDB (http://phylomedb.org) or
Saccharomyces Genome Database (SGD). Here we present a tutorial on how researchers can
access and exploit all evolutionary information contained in phylomeDB for their proteins of
interest.




                                               80
P31A
A study on parasexual cross between Candida albicans and Candida dubliniensis
Uttara Chakraborty1 and Kaustuv Sanyal2
1 Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific
Research, Jakkur, Bangalore 560064, India, Phone: +91 80 2208 2878, FAX: +918022082766, e-
mail: uttara@jncasr.ac.in
2 Molecular Mycology Laboratory,Mol Biol & Gen Unit,JNCASR,Jakkur, Bangalore 560064

Candida albicans and Candida dubliniensis are two closely related pathogenic yeasts which are
diploid and primarily asexual in nature. In spite of studies showing high degree of similarity in the
overall genome sequence and arrangement of genes in these two species, they differ largely in
having unique and different centromeric sequences on each of their eight chromosomes. Recent
studies have explained a parasexual cycle in Candida albicans and have shown that during
conjugation, two different mating type strains unite their diploid cells to form tetraploids which
then reduce back towards diploidy by a concerted chromosome loss.
In this study we have reported a parasexual cross between Candida albicans and Candida
dubliniensis by spheroplast fusion. Since these two species differ largely in their virulence
properties we were interested to study the fate of their somatic hybrids in terms of virulence as
well as genome stability. We generated stable tetraploid hybrids and obtained a single hybrid line
which retained a copy of each chromosome of both the C. albicans and C. dubliniensis parents.
A standard ChIP assay was performed to localize precisely the centromeric histone (Ca/CdCse4p)
binding sites on all the chromosomes of the hybrid and observed that the centromeres of all eight
chromosomes of each C. albicans and C. dubliniensis parents were enriched. Subcellular
immunolocalization with antiCa/CdCse4p antibody, which recognizes the CENP-A homologue,
suggested proper segregation of chromosomes in the hybrid cells. We subsequently induced
chromosome loss in this tetraploid strain and looked for diploid segregants which retained a copy
of each chromosome of both C. albicans and C. dubliniensis. Upon further investigation, we found
that the hybrid and its segregants were primarily hyper-filamentous and formed dense biofilm.
Virulence properties were assayed in a murine systemic infection model but the hybrid and its
segregants turned out to be avirulent in nature. This raises an interesting speculation as to whether
the plasticity of cellular morphology in Candida at all contributes to the virulence of this organism
or not. We are now looking forward to test the transcription profiling of the hybrid and its
segregants by microarray to see if the genes controlling the key virulence factors are differentially
regulated with respect to their parent strains or not.




                                                 81
P32B
Efg1 recruitment of NuA4 primes promoters for hypha-specific Swi/Snf binding and
activation in Candida albicans
Yang Lu1, Chang Su1, Xuming Mao1, Haoping Liu2 and Jiangye Chen1
1 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/
2 Department of Biological Chemistry, College of Medicine, University of California, Irvine,
Irvine, California 92697-1700

Efg1 is essential for hyphal development and virulence in the human pathogenic fungus Candida
albicans. How Efg1 regulates gene expression is unknown. Here, we show that Efg1 interacts with
components of the NuA4 (nucleosome acetyltransferase of H4) HAT complex in both yeast and
hyphal cells. Deleting YNG2, a subunit of the NuA4 HAT module, results in a significant
decrease in the acetylation level of nucleosomal H4 and a profound defect in hyphal development,
as well as a defect in the expression of hypha-specific genes. Using chromatin
immunoprecipitation, Efg1 and the NuA4 complex are found at the UAS regions of hypha-specific
genes in both yeast and hyphal cells, and Efg1 is required for the recruitment of NuA4.
Nucleosomal H4 acetylation at the promoters peaks during initial hyphal induction in an Efg1
dependent manner. We also find that Efg1 bound to the promoters of hypha-specific genes is
critical for recruitment of the Swi/Snf chromatin remodeling complex during hyphal induction.
Our data show that the recruitment of the NuA4 complex by Efg1 to the promoters of hypha-
specific genes is required for nucleosomal H4 acetylation at the promoters during hyphal
induction, and for subsequent binding of Swi/Snf and transcriptional activation.




                                              82
P33C
Mss11, a transcription activator, is required for hyphal development in Candida albicans
Chang Su, Yang Lu, Yandong Li, Fang Cao 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: csu@sibs.ac.cn, Web: http://www.sibs.ac.cn/

Candida albicans undergoes a morphological transition from yeast to hyphae in response to a
variety of stimuli and growth conditions. Flo8, a transcription factor containing a putative
dimerization domain named LUFS, is essential for hyphal development in C. albicans. To
determine whether the other LUFS containing factors are also involved in hyphal development of
C. albicans, we identified C. albicans Mss11, a functional homolog of S. cerevisiae Mss11. The C.
albicans Mss11 contains a LUFS domain at its N-terminus, a Q-rich domain in its central part and
an N-rich domain at its C-terminus. The Mss11 can physically and functionally interact with the
Flo8 by in vivo immunoprecipitation and reciprocal epistasis experiments. Expression levels of
MSS11 increased during hyphal induction. Overexpression of the MSS11 enhanced filamentous
growth. Deletion of the MSS11 results in a profound defect in hyphal development and expression
of hypha-specific genes. Our data suggest that the Mss11 may function as an activator, together
with the Flo8, regulate hyphal development and the expression of hypha-specific genes in C.
albicans.




                                               83
P34A
Regulation of septin dynamics through Rts1 during Candida albicans morphogenesis
David Caballero-Lima1, Alberto González-Novo2, Pilar Gutiérrez-Escribano1, Carmen Morillo-
Pantoja1, Carlos R. Vázquez de Aldana2 and Jaime Correa-Bordes1
1 Ciencias Biomédicas. Facultad de Ciencias, Universidad de Extremadura, Avda Elvas sn,
Badajoz 06071, SPAIN, Phone: +34924289300 ext 86874, FAX: +34924289300, e-mail:
jcorrea@unex.es
2 Instituto Microbiología Bioquímica. Dpto. Microbiología y Genética. CSIC/Universidad de
Salamanca

One of the characteristics of hyphal growth is the inhibition of cell separation, necessary to
maintain cell compartments attached as a filament. Recently, we have shown that the dynamics of
hyphal septin rings are essential to inhibit cell separation in hyphae and depend on the septin Sep7
and the hyphal-specific cyclin Hgc1 (MBC 19:1509-1518). Here, we describe the role of Rts1, a
regulatory subunit of the PP2A phosphatase, in septin ring assembly during yeast and hyphal
growth. In yeast cells, the fully functional Rts1-GFP protein translocates transiently from the
nucleus to the bud neck after actomyosin ring contraction, being preferentially present at the
daughter side of the septin collar. In good correlation with this asymmetry, yeast cells lacking
RTS1 fail to properly regulate septin ring division during cytokinesis. In wild-type cells, the septin
collar divides in two rings of similar diameter coincident with actomyosin ring contraction and
septum formation. In contrast, rts1 mutants have an asymmetry in septin ring diameter, and always
the ring present in the daughter cell is significantly wider than that remaining on the mother cell;
this difference normally correlates with buds having a bigger size than the mother cells. Moreover,
disassembly of septin rings when cytokinesis is finished is also compromised in rts1 mutants, and
septin rings are persistent for several cell cycles. Therefore, these results suggest that Rts1 is
involved in the control of septin ring dynamics, especially during ring splitting and ring
disassembly. Upon hyphal induction, rts1 cells give rise to a pseudohyphal-like growth.
Interestingly, septin rings were also misshapen at the division plate in these abnormal cells, and
some longitudinal septin filaments were observed at the tip of the apical cells, suggesting that Rts1
is also required for proper septin ring assembly during hyphal growth. In addition, both yeast and
hyphal cells show aberrant septin structures at different positions of the cell cortex, indicating that
Rts1 is also required for proper septin ring assembly. Together, these results indicate that Rts1 is
necessary for the normal dynamics of septin structures in all morphogenetic states of C. albicans.

This work was supported by grants BFU2006-10318 (MEC, Spain) and PRI08A017 (Junta de
Extremadura, Spain)




                                                  84
P35B
Phosphoregulation of Mob2, a LATs/NDR kinase binding partner, during morphogenesis in
Candida albicans
Pilar Gutiérrez-Escribano1, Alberto González-Novo2, David Caballero-Lima1, Carmen Pantoja-
Godoy1, Carlos R. Vázquez de Aldana2 and Jaime Correa-Bordes1
1 Ciencias Biomédicas. Facultad de Ciencias, Universidad de Extremadura, Avda Elvas sn,
Badajoz 06071, SPAIN, Phone: +34924289300 ext 86874, FAX: +34924289300, e-mail:
pilargutierrez@unex.es
2 2 Instituto Microbiología Bioquímica. Dpto. Microbiología y Genética. CSIC/Universidad de
Salamanca.

The LATs/NDR protein kinase Cbk1 and its binding partner Mob2 form a complex that is a major
downstream effector of the RAM signalling pathway. In fungi, Cbk1/Mob2 and the RAM
signalling network are important for polarised growth, differential gene expression and
maintenance of cell integrity. Recently, it has been shown that the RAM network is critically
required for hyphal growth as well as normal vegetative growth in C. albicans (MBC 2008;
19:5456-77). Here, we have used synchronous cell cultures to analyze the regulation of the fully
functional Cbk1-myc/Mob2-HA during yeast and hyphal growth. In S. cerevisiae, whereas Mob2
does not suffer any detectable post-traductional modifications throughout the cell cycle, Cbk1 is
phosphorylated in a cell cycle dependent manner (JCB 2006; 175:755-766). However, our results
suggest that phosphorylation of the Mob2 subunit is important for the regulation of the kinase
activity of the Cbk1/Mob2 complex in C. albicans. Western blot analysis of extracts from
elutriated cultures shows that Mob2 is heavily phosphorylated, with different patterns in yeast and
hyphae. During yeast growth, Mob2 is phosphorylated in a cell cycle-dependent manner at the
G1/S transition, giving rise to a clear low migrating form; in contrast, the phosphorylation pattern
in hyphae is diffuse and it is only observed at the beginning of the serum response, suggesting an
independent cell cycle phosphorylation. In order to identify the kinases that phosphorylate Mob2,
we will study the electrophoretic mobility of Mob2-HA in different backgrounds, including kic1,
ccn1, hgc1 and gin4 mutants. In this respect, Cdc28/cyclin complexes are interesting candidates to
phosphorylate Mob2, since this protein, with 313 amino acids, presents 4 CDK consensus
phosphorylation sites. To study the physiological function of the CDK phosphorylation sites, we
constructed a MOB2 allele in which serine residues were replaced with alanine at the 4 CDK
motifs and transformed it into the mob2 mutant under the control of the native promoter. We
found that mob2-4A cells appeared normal during yeast growth, but hyphae showed aberrant
morphologies. Therefore, these results indicate that the CDK consensus phosphorylation sites are
important for the Cbk1/Mob2 regulation during hyphal growth.

This work was supported by grants BFU2006-10318 (MEC, Spain) and PRI08A017 (Junta de
Extremadura, Spain)




                                                85
P36C
Forward genetics in Candida albicans that reveals ARP2 and VPS52 are required for hyphal
formation
Elias Epp1, Guylaine Lépine2, Zully Leon1, Alaka Mullick1, Martine Raymond2 and Malcolm
Whiteway1
1 Biology, McGill University, 1205 Docteur Penfield, Montréal H3A 1B1, CANADA, Phone: 001
514 496 1529, FAX: 001 514 496 6213, e-mail: elias.epp@mail.mcgill.ca
2 Institut de Recherche en Immunologie et en Cancérologie (IRIC), Université de Montréal

Genetic manipulation and functional characterization studies in C. albicans are difficult, and this
has led to application of various alternative strategies such as transcriptional profiling and the use
of surrogate models such as S. cerevisiae to link C. albicans genes to functions. To overcome the
restrictions inherent in such approaches, we have investigated a forward genetic mutagenesis
approach directly in C. albicans. We screened 4700 random insertion mutants for defects in
hyphal development, and identified known as well as new genes linked to hyphal growth.
Mutations in VPS52 and ARP2 led to significant reductions in hyphal formation. The arp2 and
arp2/arp3 double mutants were found to share many, but not all, phenotypes with C. albicans
mutants for myo5 or wal1, two Arp2/3 complex activators. vps52 mutants showed a typical VPS
class B fragmented vacuolar phenotype. Both arp2 and vps52 mutants were significantly reduced
in virulence in a mouse-tail vein model of disseminated candidiasis. Repeating our screening
approach for mutants unable to grow on glycerol identified an insertion in ORF19.875, which has
no obvious homolog in other well-studied fungi. This forward genetic approach allows linking a
function to a gene directly in Candida albicans and therefore should help in defining C. albicans-
specific traits to better understand how they contribute to the lifestyle of this medically important
fungus.




                                                 86
P37A
Re-wiring of the Bcr1 transcription factor in C. albicans and C. parapsilosis
Chen Ding, Alessandro Guida, John Synnott, Leona Connolly and Geraldine Butler
School of Biomolecular and Biomedical Science, Conway Institute, Belfield, Dublin NA, Ireland,
Phone: +3531716 6841, FAX: +35312837211, e-mail: chen.ding@ucd.ie

Bcr1 is an important regulator of biofilm formation in both Candida albicans and Candida
parapsilosis. In C. albicans, Bcr1 regulates the expression of potential adhesins and cell wall
genes, including ALS1, ALS3, HWP1 and RBT5. Here, we compare the transcriptional profile of
bcr1 knockouts in the two species. We show that there is surprisingly little overlap in the targets;
we found 10 genes that are regulated in both species, of which only 6 genes have the same
regulation pattern.
One common feature is that Bcr1 regulates expression of several members of the CFEM family
(Common in Several Fungal Extracellular Membrane proteins) in both C. albicans and C.
parapsilosis. In C. albicans, there are 5 CFEM members (CSA1, CSA2, RBT5, PGA7, and
PGA10), several of which are important for biofilm development. Expression of RBT5 and PGA7
is regulated by Bcr1. The CFEM family has undergone an expansion to 7 members in C.
parapsilosis, which we call CFEM1-7. There are two paralogues of each of CSA1 (CFEM6,
CFEM7), RBT5 (CFEM1, CFEM2) and PGA7 (CFEM3, CFEM4), and expression of one of each
gene pair (CFEM2, CFEM3 and CFEM6) is reduced from 6- to 20-fold in a bcr1 deletion.
In C. albicans Rbt5 and Pga10 are required for iron acquisition from haem. We show that when
CFEM2 and CFEM3 are deleted C. parapsilosis loses the ability to utilise haem as a sole iron
source. Haem utilisation is restored when CFEM3 is re-integrated.
We also noticed that expression of three genes associated with iron transport (CFL5, FTR1, and
FRP1) are down-regulated in a Cpbcr1Δ;, but not in C. albicans. This led us to investigate the
utilisation of iron in C. parapsilosis. We determined the transcriptional profile of C. parapsilosis
in low iron conditions, and show that several genes have differential expression in iron-depleted
conditions and are that are regulated by Bcr1, including CFEM family members (CFEM2, CFEM3
and CFEM6), and a iron permease FTR1 and the ferric reductase FRP1. We also show that Bcr1 is
required for induction of expression of some of the CFEM family by iron limitation.
Our results suggest that there has been some conservation of function in the Bcr1 pathway
between C. albicans and C. parapsilosis, but there is also evidence for substantial re-wiring. We
have generated knockouts of other targets of Bcr1 in C. parapsilosis, and are currently
investigating their role in biofilm development.




                                                87
P38B
Rapid detection and quantification of Aspergillus fumigatus in air using solid-phase
cytometry
Lies Vanhee, Hans Nelis and Tom Coenye
Ghent University, Laboratory of Pharmaceutical Microbiology, Harelbekestraat 72, Ghent 9000,
Belgium, Phone: +32 9 264 8142, FAX: +32 9 264 8195, e-mail: Lies.Vanhee@UGent.be

A. fumigatus is an ubiquitous fungus causing severe infections such as aspergilloma, allergic
bronchopulmonary aspergillosis and invasive aspergillosis in immunocompromised patients.
Monitoring of the number of A. fumigatus spores in the air inhaled by these patients is crucial for
infection control. In the present study, a new and rapid technique for the quantification of A.
fumigatus, based on solid-phase cytometry and immunofluoresent labelling, has been developed.
Air samples were collected by impaction on a water soluble polymer that was subsequentely
dissolved. A part of the sample was filtered and microcolonies were allowed to form on the filter
for 18 hours at 47°C. Subsequentely, labelling with a monoclonal anti-Aspergillus antibody and
tyramide signal amplification was used to detect the microcolonies with the aid of a solid phase
cytometer (ChemScan RDI). The detected spots were microscopically validated using an
epifluorescence microscope. The sensitivity and specificity of the assay were evaluated by testing
pure cultures of 40 A. fumigatus strains, 12 other Aspergillus species, 14 different Penicillium
species and 14 other filamentous fungi. All A. fumigatus strains yielded labelled microcolonies,
which confirmed the sensitivity of the assay. Only Rhizopus stolonifer and Paecilomyces variotii
were labelled with the antibody and were able to form microcolonies at 47°C. These fungi,
however, could be discriminated from A. fumigatus based on morphology. Comparison with
traditional culture-based methods indicated that our novel approach is a rapid and reliable
alternative with a high dynamic range.




                                                88
P39C
Volatile communication in the human pathogen Candida albicans
Rebecca Hall1, Rebecca Eaton1, Clemens Steegborn2 and Fritz A. Muhlschlegel1
1 Department of Biosciences, University of Kent, Giles Lane, Canterbury CT2 7NJ, UK, Phone:
+44 (0) 1227 823735, FAX: +44 (0)1227 763912, e-mail: r.a.hall@kent.ac.uk, Web:
http://www.kent.ac.uk/bio/muhlschlegel/
2 Department of Physiological Chemistry, Ruhr-University Bochum, Germany

Host environmental factors including serum and pH are known to regulate C. albicans
morphology and thus virulence. Furthermore, C. albicans cells generating fungal biomasses, such
as those found in superficial epithelial infections or biofilms, are exposed to signal gradients.
Indeed, cells in the centre of the colony will be confronted with different conditions to those at the
periphery. As greater biomasses are established, the diffusion of volatile molecules becomes
limited. The concentrations of specific gaseous molecules are known to influence C. albicans
morphology, with carbon dioxide being a classical example of this. We found that when grown
under diffusion limiting conditions, C. albicans SC5314 displayed variations in colony
morphology, which were dependent on cell density. In contrast, diffusion permitting conditions
only promoted the growth of yeast colonies. Colony phenotypes were dependent on cAMP, as the
adenylyl cyclase (cyr1) mutant retained yeast colony growth under all conditions. As Cyr1 is
directly activated by CO2/bicarbonate ions, the carbonic anhydrase (nce103) mutant, which is a
sensitive bioindicator of CO2 levels, was used to measure the CO2 concentration. Diffusion
limiting conditions promoted the growth of the nce103 mutant, suggesting an accumulation of
self-generated CO2. Selective removal of CO2 from the head space, by the addition of a NaOH
trap, inhibited growth of the nce103 mutant and colony morphology of SC5314. Furthermore,
growth of the nce103 mutant was sustained when grown in a single, mixed colony, confirming
that CO2 gradients occur at colony level. Biomass-associated phenotypes are under investigated in
C. albicans, but could lead to an enhanced understanding, and thus better management/treatment
of fungal infections. The role of intra (and inter) colony gaseous gradients in C. albicans
morphology changes and virulence are discussed.




                                                 89
P40A
Sensory perception in pathogenic fungi: application of the split-ubiquitin membrane yeast
two-hybrid system to engineer misappropriation of response in Aspergillus fumigatus
Margherita Bertuzzi1, Maria Jimena Gonzales Gonzales1, Igor Stagljar2 and Elaine Bignell1
1 Dept. of Microbiology, Imperial College London, Armstrong Road, London UK SW7 2AZ, UK,
Phone:      +44(0)20      7594     5293,    FAX:       +44(0)20     7594     3095,      e-mail:
margherita.bertuzzi06@imperial.ac.uk
2 b. Dept. of Biochemistry & Dept. of Medical Genetics and Microbiology, University of Toronto

Environmental adaptation is of paramount importance to all microbes, especially those inhabiting
mammalian niches during infection. Protein interactions at the plasma membrane are formative
events in environmental sensing and may provide a means to prevent appropriate host-adaptation.
Adaptation to environmental pH is crucial for Aspergillus virulence. Normal functioning of this
regulatory system in the model ascomycete A. nidulans requires the integrity of seven genes. PacC
is a DNA binding transcription factor for genes required for growth at alkaline ambient pH.
Ambient signal transduction for PacC proteolytic activation involves a further six proteins, PalA,
B, C, F and I. pH signalling connects two protein complexes, the first plasma membrane-localised
and composed of two putative pH signal receptors, PalH and PalI, plus a cytoplasmic non-
metazoan member of the arrestin family, PalF.
As demonstrated in both A. nidulans, and the pathogen Aspergillus fumigatus, deletion of PacC,
thereby blocking ambient pH signal transduction strongly attenuates virulence in neutropenic
mice. Sensory deprivation could therefore provide a means to inhibit infectious fungal growth. In
the absence of either PalH or PalI pH signalling is blocked and PacC is not processed.
Currently we are using the Saccharomyces cerevisiae membrane yeast two-hybrid (MYTH)
system to test the hypothesis that A. fumigatus PalH and PalI interact in the plasma membrane of
the cells, and that abolishing the interaction attenuates virulence. Using a membrane-associated
protein for the screening, this technique allows the isolation of novel protein interactors from a
full-length A. fumigatus cDNA library. The split-ubiquitin screen will permit identification of
novel interacting partners of PalH and PalI, whose roles in virulence could be tested by deletion in
A. fumigatus. Thus, mechanisms non-essential for A. fumigatus viability, but crucial for its
virulence, could provide new opportunities to prevent fungal growth in vivo.
Bignell, E, et al., (2005), Mol Microbiol, 55(4), 1072
Fetchko, M, et al., (2004), Methods, 32(4), 349
Peñalva, MA, et al., (2008), Trends Microbiol, 16(6), 291




                                                90
P41B
The conservation of a heat shock response in an obligate pathogen of warm-blooded animals
Michelle Leach, Susan Nicholls and Alistair JP Brown
Aberdeen Fungal Group, University of Aberdeen, Institute of Medical Sciences, Foresterhill,
Aberdeen AB25 2ZD, UK, Phone: +44 (0)1224 555883, FAX: +44 (0)1224 555844, e-mail:
michelle.leach@abdn.ac.uk

The major fungal pathogen of humans, Candida albicans is thought to be obligately associated
with warm-blooded mammals. In this niche the fungus is unlikely to be exposed to sudden and
dramatic changes in temperature, yet C. albicans has retained a heat shock response. The classic
heat shock response is defined by the up-regulation of heat shock genes and proteins, many of
which are chaperones involved in protein (re)folding, as a result of a sudden up-shift in
temperature in vitro. We have shown that a functional heat shock regulon, the conserved heat
shock transcription factor (Hsf1), is retained in C. albicans. It activates the transcription of target
genes via classic heat shock elements in their promoters. Furthermore, our microarray studies have
confirmed that in C. albicans, Hsf1 contributes to the global transcriptional response to heat shock
induction. But why should this response be conserved in C. albicans? Of course it is not possible
to exclude the possibility that C. albicans inhabits as yet undefined environmental niches where it
is exposed to a heat shock. Alternatively, Hsf1 might be essential for responses to other medically-
relevant stresses. However, our studies suggest that this is not the case. Instead, Hsf1 may be
required for the expression of essential functions under non-stress conditions. Key chaperone
genes in C. albicans depend on Hsf1 for their expression under normal conditions. Furthermore in
the absence of a heat shock, Hsf1 appears to tune the expression of HSE-containing genes to the
growth temperature of C. albicans cells. This supports the notion that Hsf1 in C. albicans might
be the homeostatic regulator of chaperone levels in response to growth temperature. Therefore, the
Hsf1-HSE regulon appears to act as a temperature rheostat, in addition to a heat shock switch.




                                                  91
P42C
Analysis of chlamydospore production by Candida albicans and Candida dubliniensis
Francesco Citiulo, Gary Moran, David Coleman and Derek Sullivan
Oral Microbiology, Dublin Dental school and Hospital, Lincoln place, Dublin ABC123, Ireland,
Phone: +353857067837, FAX: 0035316127295, e-mail: francesco.citiulo@dental.tcd.ie

Candida albicans and Candida dubliniensis are the only members of the Candida genus with the
ability to produce chlamydospores. The function of these cells is not currently known and their
analysis has been hampered by difficulties in culturing them in vitro. We have previously reported
the development of novel conditions that induce the hyperproduction of chlamydospores by both
species in liquid media. This has allowed us to purify these structures and investigate their
biology. Staining of attached and purified chlamydospores with the metabolic molecular probe
FUN-1 revealed that young chlamydospores are metabolically active, but this activity decreases
over time (i.e. following 15-20 days incubation), this is in contrast to blastospores, which
maintained metabolic activity during the same time period. Transcriptomic analysis revealed that
specific genes involved in respiration, cell cycle check points and chromatin condensation are
differentially regulated in chlamydospores and yeast cells (eg. MRP2, MEC1, MAD2, CBF1,
SMC2). Inactive chlamydospores were phagocytosed and killed by murine RAW 264.7
macrophages. Addition of nutrients and serum to metabolically inactive chlamydospores resulted
in a gradual resumption of metabolic activity, followed by, depending on the conditions, budding,
pseudohypa or hypha formation which was observed using light and scanning electron
microscopy. When chlamydospores were preincubated in serum they were observed to germinate
and escape from macrophages. Although there have been very few reports of the production of
chlamydospores in vivo, we have observed chlamydospore-like structures in electron micrographs
of Galleria melonella larvae that were infected with a lethal dose of C. albicans blastospores, 8 to
20 days following death of the larva. These data suggest that these cells may play a role in the
normal life cycle of C. albicans and C. dubliniensis.




                                                92
P43A
Targeted protein aggregation as a new tool for protein inactivation in Candida albicans
Alessandro Fiori and Patrick Van Dijck
VIB Department of Molecular Microbiology, Katholieke Universiteit Leuven, Kasteelpark
Arenberg 31, Heverlee 3001, Belgium, Phone: +32-(0)16320368, FAX: +32 (0)16321979, e-mail:
alessandro.fiori@mmbio.vib-kuleuven.be

Candida albicans is the most common human fungal pathogen. Infections caused by this
dimorphic fungus range from superficial to life-threatening, systemic mycoses in
immunocompromised individuals. Treatments against C. albicans with commonly available
antifungals are hampered by the frequent onset of resistance of the fungus or by intrinsic toxicity
of the drugs (amphotericin B).
On the other hand, conversion of soluble proteins into amorphous aggregates and amyloid fibrils
is a major issue in today’s biology and medicine. Spontaneous aggregation appears to be subject to
structurally determined mechanisms, such as enrichment in hydrophobic sequences with beta-
structure content (Rousseau et al., 2006).
We intend to test the possibility to induce in vivo aggregation of fungal-specific proteins, in order
to control the growth and/or pathogenicity of C. albicans. Aggregation of the target proteins
should produce a phenotype similar to that of the corresponding deletion mutants.
In order to induce targeted aggregation of C. albicans Gsc1p/Fks1p (Mio et al., 1997), we scanned
its amino acid sequence using TANGO, a statistical mechanics algorithm for prediction of
peptides’ propensity to aggregation. A 17-mer peptide was selected and tested for its ability to
trigger aggregation of the corresponding native proteins upon overexpression. Expression of such
peptide fused to Gfp (aggregator), driven from the PCK1 inducible promoter, caused a dramatic,
reversible, negative effect on growth of some of the transformants. The aggregator accumulated in
the perinuclear endoplasmic reticulum of transformants and aggregated in a detergent-insoluble
fashion. The corresponding cells appeared irregular, not separated after budding, and sometimes in
a tree-like structure similar to that observed in other mutants with cell wall defects. In addition,
their lethality could be rescued by the addition of sorbitol to the growth medium, suggesting that
the lethal phenotype arises from severe damage to their cell wall caused by depletion of functional
Gsc1p.
Taken together, our experiments demonstrate that targeted protein aggregation has the potential to
become a technology platform for protein knock-out in C. albicans, and possibly in other
pathogenic fungi, even though further experiments are needed to fine-tune the system.
Mio, T., et al. (1997). J Bacteriol 179, 4096.
Rousseau, et al. (2006). J Mol Biol 355, 1037.




                                                 93
P44B
Transcriptional loops meet chromatin: a dual-layer network controls white-opaque
switching in C. albicans
Denes Hnisz, Tobias Schwarzmueller, Walter Glaser and Karl Kuchler
Christian Doppler Laboratory for Infection Biology, Medical University Vienna, Dr. Bohr-Gasse
9/2, Vienna A-1030, Austria, Phone: +43-1-4277-61807, FAX: +43-1-4277-9618, e-mail:
karl.kuchler@meduniwien.ac.at

Candida albicans is a diploid opportunistic human fungal pathogen causing superficial and
systemic infections. The phenotypic plasticity that allows C. albicans to adapt to various host
niches is considered a major virulence attribute. C. albicans can exist as a unicellular yeast, but
also able to form pseudo- as well as real hyphae morphologies. Furthermore, C. albicans is able to
undergo a reversible switch between two distinct cell morphologies called white and opaque,
which are considered as different transcriptional states of cells harboring identical genomes. The
present model of switching regulation includes the bistable expression of a master switch gene that
is controlled by multiple transcriptional feedback loops. The major goal of our studies was to
identify novel histone-modifying enzymes implicated in white-opaque switching in C. albicans
Hence, we constructed homozygous deletion mutants all putative histone-modifying genes in a
background homozygous for the MTL locus. We then assayed the impact of gene deletions on the
white-opaque and on the opaque-white switching frequencies. Here, we show that chromatin-
modifying enzymes constitute an additional regulatory layer of switching. We identify chromatin
modifiers as novel switching modulators. Epistasis analysis mapped the genes into at least two
distinct pathways, some of which overlay the known transcriptional network. The conserved
Set3/Hos2 histone deacetylase complex was identified as a key regulator that relies on the
methylation status of lysine 4 on histone H3 in switching modulation. We propose that chromatin
modifications may integrate environmental or host-derived stimuli through underlying
transcriptional circuits to determine cell fate in C. albicans
This work was supported by a grant from the Christian Doppler Research Society, and the
transnational ERA-Net Pathogenomics project FunPath (Austrian Science Foundation FWF-I125-
B09). DH is a Vienna Biocenter PhD Student Fellow.




                                                94
P45C
Intracellular proteinases Apr1p and Cpy1p from Candida albicans
Vaclava Bauerova, Elena Dolejsi, Olga Hruskova-Heidingsfeldova and Iva Pichova
Institute of Organic Chemistry and Biochemistry, Flemingovo n.2, Prague 16610, Czech republic,
Phone: +420 220 183 242, FAX: +420 220 183 556, e-mail: vaclava@uochb.cas.cz, Web:
http://www.iocb.cz

Vacuoles of Candida albicans undergo significant morphological changes during the culture
growth. Changes in vacuolar size might indicate the importance of vacuolar hydrolysis in
metabolic adaptation to varying environmental conditions such as starvation induced by Candida’s
encapsulation by macrophage or adaptation to stress conditions. Due to possible participation of
vacuole metabolism in the survival of this opportunistic pathogen, vacuolar proteinases deserve
attention.
We have partially purified aspartic protease Apr1p from C. albicans cells and determined its N-
terminal sequence. For detection of Apr1p during the course of isolation, we have used polyclonal
hen’s antibodies prepared against peptides derived from the Apr1p sequence. Localization of
Apr1p in vacuoles was proven by isolation of vacuoles and analysis by western blots and an
activity assay using chromogenic substrate KPAEFF(NO2)AL. Proteinase in vacuolar fraction
was found to be active and was inhibited by pepstatin A, which confirms, that the activity can be
really attributed to the aspartic proteinase.
Carboxypeptidase Y is known to be a typical vacuolar marker in S. cerevisiae, therefore we
analyzed the vacuolar fractions from C. albicans for the presence of carboxypeptidase Cpy1p. We
have found that the commercial polyclonal rabbit antibodies against CpY from S. cerevisiae cross-
react with Cpy1p from C. albicans. Also the Cpy1p N-terminal sequence was determined.
During the culture growth Cpy1p highly exceeds the amount of Apr1p as we observed on western
blots and SDS PAGE. In the early stage of exponential growth, there is no detectable Cpy1p or
Apr1p on western blots, but as the culture growth proceeds to the late exponential stage, the
amount of Cpy1p and Apr1p increase. In the late stationary phase the amount of these proteins
again drops. Since we wanted to follow not only the translational but also their transcriptional
development, qPCR was performed. The detailed analysis of gene and protein expression under
varying conditions, such as starvation, will enable us to elucidate the role of these enzymes in C.
albicans. This work was supported by the Czech Science Foundation (grant 310/09/1945) and by
the Ministry of Education of the Czech republic (grant LC 531).




                                                95
P46A
Alkali metal cation transporters involved in salt tolerance of Candida glabrata
Yannick Krauke 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

Candida physiology and pathogenicity depends on and is influenced by many factors. Among
them, the internal/external pH and alkali-metal-cation concentrations play an important role. Cells
must maintain optimally high intracellular concentration of potassium and low concentration of
toxic sodium. Yeast species usually possess two types of systems to export surplus alkali metal
cations, Na+-ATPases and Na+/H+ antiporters. Our in silico search revealed the existence of
corresponding genes in all Candida species. C. glabrata is more tolerant to alkali metal cations
than S. cerevisiae, but its genome contains only one copy of genes encoding the putative ATPase
(CgENA1) and antiporter (CgCNH1). To assess the role of these two putative transporters in C.
glabrata tolerance to salts, the knock-out mutant strains lacking one or both genes have been
constructed in the ATCC2001 wild type and their phenotypes tested.
All mutants were viable, the cnh1 mutant showed a reduced tolerance to high external
concentration of K+ but not to Na+. On the contrary, the ena1 deletion strain was sensitive to Na+
but not to K+. The ena1cnh1 mutant was sensitive to all tested salts. Cation efflux measurements
confirmed the diminished ability of cnh1 cells to export potassium. Both cnh1 and ena1 mutants
showed a reduced efflux of Na+, nevertheless, the decrease in Na+ efflux was more substantial in
ena1 cells. The observed mutant phenotypes were confirmed by reintegration of corresponding
genes to the C. glabrata genome. Obtained results suggest that the role of Nha1/Cnh1 antiporters
and Ena ATPases is different in S. cerevisiae and C. glabrata. In S. cerevisiae, both transporters
cooperate and are involved in efflux of Na+ and K+, whereas in C. glabrata they seem to fulfill
more specialized roles, CgCnh1p being important for potassium homeostasis and CgEna1p for
sodium detoxification.

This work was supported by MRTN-CT-2004-512481 and MSMT LC531.




                                                96
P47B
How do neutrophils detect, inhibit and kill Candida albicans?
Pedro Miramón Martínez, Antje Albrecht, Ines Leonhardt and Bernhard Hube
Microbial Pathogenicity Mechanisms, Hans Knoell Institute, Beutenbergstrasse 11a, Jena 07745,
Germany, Phone: +49 (0) 3641 532 0359, FAX: +49 (0) 3641 532 0810, e-mail:
pedro.miramon@hki-jena.de, Web: http://www.hki-jena.de/index.php

Candida albicans can disseminate in the host via the bloodstream and cause life-threatening
systemic infections. Yet, this is surprising as blood is generally sterile and represents a hostile
environment for all microorganisms. Our previous work has shown that blood components
influence the morphology, viability and transcriptional response of C. albicans (Fradin et al.,
(2003) Mol Microbiol 47: 1523-43.; Fradin et al., (2005) Mol Microbiol 56: 397-415). Of these,
neutrophils have the strongest effect. However, the mechanisms by which neutrophils detect,
inhibit and kill C. albicans remain unclear. When C. albicans cells were incubated with purified
neutrophils, we found that genes associated with detoxification of reactive oxygen species, genes
encoding enzymes of the glyoxylate cycle and genes associated with amino acid metabolism were
all upregulated. However, the expression pattern reflected the sum of different populations of C.
albicans cells, for example, those cells which were attached to, phagocytosed by or not in contact
with neutrophils. Furthermore, hyphae, but not yeast cells induced targeted motility of neutrophils
towards the fungus (Wozniok et al. (2008) Cell Microbiol 10: 807-20). Although only hyphae
were in direct contact with neutrophils, both morphological forms were killed. Therefore, we
propose that the recognition and killing mechanisms of neutrophils differ between yeast and
hyphae.
In order to elucidate the mechanisms by which neutrophils detect, inhibit and kill C. albicans,
ongoing experiments concentrate on (a) the analysis of single cells exposed to neutrophils using
GFP reporter strains, (b) the interaction of yeast and hyphal cells of wild type and (c) mutants
strains with neutrophils and (d) the role of the C. albicans cell surface associated antigens such as
Pra1 for attraction of neutrophils. Current data from these experiments will be presented.




                                                 97
P48C
Genome-wide identification of transcriptional regulators of morphogenetic variability in
Candida albicans
Michael Weyler, Tina Schüll and Joachim Morschhäuser
Institut für Molekulare Infektionsbiologie, Röntgenring 11, Würzburg 97070, Germany, Phone:
+49 931 312127, FAX: +49 931 312578, e-mail: Michael.Weyler@uni-wuerzburg.de

The human fungal pathogen Candida albicans displays a remarkable morphogenetic variability. In
addition to growing as a budding yeast, C. albicans can also grow as filaments (hyphae and
pseudohyphae) or form chlamydospores, depending on the environmental conditions. Strains that
are homozygous at the mating type locus (MTL) can also switch from the normal yeast form
(white) to an elongated cell type (opaque), which is the mating competent form of the fungus.
Transcription factors play an important role in the regulation of all these developmental processes,
and the expression of the regulators themselves is often modulated during morphogenesis.
Therefore, morphogenesis can be stimulated under normally noninducing conditions by forced
expression of activators or by downregulation of repressors. To identify novel regulators of
filamentous growth, chlamydospore formation, and white-opaque switching, we have cloned more
than 300 genes encoding confirmed or putative C. albicans transcription factors (Arnaud, MB, et
al., (2007), N.A.R., 35, 452;www.candidagenome.org), including known hyperactive alleles of
some of these genes, and placed them under the control of a tetracycline-inducible promoter (Park,
YN, et al., (2005), E.C. , 4, 1328). The inducible gene expression cassettes were introduced into
the genomes of the C. albicans model strain SC5314 and the white-opaque switching-competent
MTLalpha strain WO-1. The libraries are currently screened by growing the strains in the presence
of doxycycline to induce gene expression under different conditions and identify genes that induce
or inhibit morphogenesis. The results of these screenings and the functional analysis of the
identified regulators will be presented.




                                                98
P49A
Systematic analysis of kinase and phosphatase function in Candida albicans’ yeast to hyphae
transition
Christian Schmauch1, Bernardo Ramírez-Zavala2, Tsvia Gildor3, Daniel Kornitzer3, Joachim
Morschhäuser2 and Robert Arkowitz1
1 Institute of Developmental Biology and Cancer, CNRS UMR6543, Université Nice - Sophia
Antipolis, Parc Valrose, Nice 06108, France, Phone: +33 (0)4 9207 6465, FAX: +33 (0)4 9207
6466, e-mail: schmauch@unice.fr, Web: www.unice.fr/isdbc/
2 Institut für Molekulare Infektionsbiologie, Universität Würzburg, Würzburg, Germany
3 Department of Molecular Microbiology, B. Rappaport Faculty of Medicine, Haifa, Israel

An important factor in the pathogenicity of Candida albicans is its ability to exhibit a large
morphological variability in response to changing environmental conditions. In particular, the
morphogenetic switch between the yeast and hyphal form is thought to be an important virulence
trait, helping the organism to gain access to and to proliferate in new host niches. To elucidate new
genes that regulate this yeast to hyphae transition we systematically analyzed all identifiable
protein kinases, phosphatases and their regulators in this morphogenetic process [1].
We have used an inducible expression strain library to identify proteins that, when expressed,
promote or inhibit the yeast to hyphal transition. To generate this library every gene was cloned
into a tetracycline inducible promoter system [2] and fully sequenced. These constructs were than
each integrated into the genome of strain SC5314 and confirmed by southern blot analysis. The
resulting library comprises a total of 224 strains, covering 123 verified and putative kinases, 39
phosphatases, 25 kinase and 6 phosphatase regulators. In addition to these wildtype genes, >30
mutant alleles were generated.
After screening two independent clones of each strain we have initially identified 22 different
proteins that affect the process of filamentation. Confirming the validity of this approach, among
these 22 were 13 proteins that have been previously shown to be involved in hyphal
morphogenesis, including members of different MAP kinase cascades and cell cycle regulators. In
addition to these previously characterized genes, our screen has identified nine genes whose role
in C. albicans filamentation has not been described. Currently, we are examining the molecular
functions of these proteins in the yeast to hyphal transition and whether they function in
previously described pathways or define novel pathways. This work is supported by ERA-NET
PathoGenoMics.

References:
1. Arnaud MB, Costanzo MC, Skrzypek MS, Shah P, Binkley G, Lane C, Miyasato SR, and
Sherlock G, "Candida Genome Database", http://www.candidagenome.org/
2. Park, YN and Morschhäuser J, (2005), Eukaryot Cell, 4, 1328




                                                 99
P50B
White-opaque switching in Candida albicans is regulated by protein kinase signalling
Bernardo Ramirez-Zavala1, Christian Schmauch2, Tsvia Gildor3, Daniel Kornitzer3, Robert
Arkowitz2 and Joachim Morschhäuser1
1 Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg
97070, Germany, Phone: +0049 931 312127, FAX: +0049 931 312578, e-mail: b.ramirez@uni-
wuerzburg.de
2 Institute of Developmental Biology and Cancer, CNRS UMR6543, Université Nice - Sophia
Antipolis, Nice, France
3 Department of Molecular Microbiology, B. Rappaport Faculty of Medicine, Haifa ,Israel

Candida albicans strains that are homozygous at the mating type locus can reversibly switch from
the normal yeast form (white) to an elongated cell form (opaque), which is the mating competent
form of the fungus. The two cell types also differ in their ability to colonize and infect various
tissues; therefore, switching may allow C. albicans a better adaptation to different host niches.
White-opaque switching occurs spontaneously at a low frequency and is controlled by several
positively and negatively acting transcription factors, which form a transcriptional feedback loop
that ensures the semi-stable maintenance of the two phases. However, white-opaque switching can
also be induced by environmental signals, indicating that upstream regulators may control the
activity of these transcription factors in response to such signals. In order to systematically search
for such regulators, we expressed all putative protein kinases, phosphatases, and their regulators
(123 kinases, 25 kinase regulators, 39 phosphatases, and 6 phosphatase regulators, which were
identified in the C. albicans genome [1]), as well as >30 predicted hyperactive or dominant-
negative alleles of these genes from a tetracycline-inducible promoter [2] in the MTLa strain WO-
1. Screening of this library of strains identified three protein kinases whose forced expression
efficiently stimulated white cells to switch to the opaque phase. Results of the functional analysis
of these kinases, their relationship to the known regulators of white-opaque switching, and their
importance in the transduction of environmental signals that influence switching will be presented.

This work was supported by ERA-NET PathoGenoMics.

References
1. Arnaud, MB, et al., (2007), Nucleic Acids Res., 35, 452; http://www.candidagenome.org/
2. Park, YN, et al., (2005), Eukaryot. Cell, 4, 1328




                                                 100
P51C
Localization studies of the moonlighting protein Tsa1p in C. albicans
Martina Brachhold1, David M. Arana2, Jesus Pla2 and Steffen Rupp1
1 Molecular Biotechnology, Fraunhofer IGB, Nobelstrasse 12, Stuttgart 70569, GERMANY,
Phone:    +49    (0)711     970    4145,     FAX:    +49     (0)711    970   4200,    e-mail:
Martina.Brachhold@igb.fraunhofer.de, Web: www.igb.fraunhofer.de
2 Departamento de Microbiologia II , Facultad de Farmacia, Universidad Complutense de Madrid,
Plaza de Ramon y Cajal s/n, E-28040 Madrid, Spain

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 (Thiol-specific antioxidant-like protein) as
part of the cell wall and within the cytoplasm of C. albicans. Tsa1p has been shown to be
responsible for several distinct functions, including functions in oxidative stress and genome
stability. It 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.
We could show that localization of Tsa1p to the cell wall is determined by at least two different
parameters. In previous experiments Tsa1p could only be detected at the cell surface in hyphae-
inducing media indicating a morphology-dependent localization of Tsa1p to the cell surface. In
addition, time course experiments showed that transfer to fresh media components also induces a
temporary translocation of Tsa1p to the cell surface in yeast form cells. This indicates a
connection with quorum sensing. Indeed, addition of farnesol to YPD medium results in stronger
and longer lasting accumulation of Tsa1p to the cell surface.
To check on regions within TSA1 that are required for localization, we deleted the C-terminal 12
amino acids of CaTSA1. From its human homologue it is known that a signal for membrane
localization resides in the C-terminal part. Additionally, the cysteines of the two active sites of
Tsa1p were substituted by serines to check their role in Tsa1p function and localization.
All mutants showed sensitivity to oxidative stress (H2O2) like the delta-TSA1 strain. Cell surface
localization of Tsa1p in the active site mutants is strongly reduced compared to the wildtype
indicating that an active form of Tsa1p is needed for localization of Tsa1p to the cell surface.
However, in the mutant strain containing the TSA1 copy with deleted C-terminus Tsa1p is still
able to localize to the cell surface, indicating that the C-terminus is not responsible for its
localization in C. albicans. In addition, all mutants show a reduced survival rate when exposed to
human neutrophils and also have a higher beta-glucan exposure at the cell surface of blastospores
compared to the wildtype. This confirms that the cell wall composition in these mutants is altered
and that Tsa1p has an important role in maintaining the cell wall composition.




                                                 101
P52A
Characterisation of a putative regulator of secreted proteases in the human pathogen A.
fumigatus
Anna Bergmann1, Thomas Hartmann1, Elaine Bignell2 and Sven Krappmann1
1 , Research Center for Infectious Diseases, Roentgenring 11, Wuerzburg 97070, Germany,
Phone: +49-931-31-2125, FAX: +49-931-312578, e-mail: Anna.Bergmann@uni-wuerzburg.de
2 Department of Microbiology, Imperial College London, London, United Kingdom

As a saprophte, the air-borne pathogen A. fumigatus is well adapted to feed from the environment
by degradation of polymeric substances and uptake of breakdown products. Correspondingly, its
nutritional versatility has to be regarded a virulence determinant in the onset of pulmonary
aspergillosis, and extracellular proteolytic activities that degrade the surrounding tissue to receive
proteinaceous nutrients may contribute to pathogenicity. The prtT gene product, first characterised
in Aspergillus oryzae, appears to be a global regulator of extracellular proteolytic activity and is
therefore involved in degradation of polymeric substances from the environment. We identified
the orthologue in the human pathogen A. fumigatus via alignment searches, and deletion of the
prtT gene, which encodes a fungus-specific zinc-cluster protein, results in a strain unable to grow
on medium with BSA or casein as sole sources of nitrogen. Furthermore, we were able to show
that the extracellular proteolytic activity of the deletant is strongly decreased, which was
substantiated by determining the transcription profiles of selected protease genes. Accordingly,
first data on a global regulator of A. fumigatus proteolytic activities will be presented to address
the role of extracellular proteases in nutritional versatility and virulence of this fungal pathogen.




                                                 102
P53B
Characterisation of the Saccharomyces cerevisiae cell separation machinery
Hsueh-lui Ho1, Marissa Vignali2, Lara West1, Helen Findon1, Florencia Minuzzi1, Stanley Fields2
and Ken Haynes1
1 Department of Microbiology, Imperial College London, 5.40 Armstrong Road, London SW7
2AZ, United Kingdom, Phone: +44 02075947409, FAX: +44 02075943095, e-mail: hsueh-
lui.ho04@imperial.ac.uk, Web: http://www3.imperial.ac.uk/cmmi
2 Department of Genome Sciences, Foege Building, University of Washington, Seattle,
Washington, USA

Previous studies have demonstrated that infection with Candida glabrata ace2 and cts1 cells in a
murine model of systemic candidiasis resulted in a hypervirulent phenotype. Both C. glabrata
ace2 and cts1 cells have a cell separation defect.
In Saccharomyces cerevisiae, Ace2 plays a central role in cell separation by regulating daughter
cell specific expression of endochitinase (CTS1) and at least 3 putative glucanase encoding genes,
DSE2, DSE4 (ENG1) and SCW11. The products of these genes degrade the tri-laminar septum
that holds mother and daughter cells together. ACE2, itself, is regulated by the RAM (Regulation
of Ace2 activity and cellular Morphogenesis) network; inactivation of RAM network proteins
results in defects in cell separation and mis-localisation of Ace2. It is possible that other mutations
that result in a cell separation defect may impact on the ace2 and cts1 hypervirulent phenotype
seen in C. glabrata.
A screen of the S. cerevisiae Yeast Knockout library and Tetracycline repressible library for
mutants that had a cell separation defective phenotype was undertaken to identify putative genes
involved in the cell separation machinery. A total of 178 novel cell separation defective mutants
were identified. Furthermore, the screen indicated that genes involved in ubquitination and
glycosylation may play a role in cell separation. Subsequent studies have shown that C. glabrata
anp1, mnn2, mnn4, and mnn6 cells have a hypervirulent phenotype in a murine model of systemic
candidiasis; ANP1, MNN2, MNN4, and MNN6 are involved in N-glycosylation in S. cerevisiae.
The uncharacterised gene, YIR016W, termed Defective in Separation of Daughter and Mother
Cell 1 (SDM1), is proposed to play an important role in cell separation in S. cerevisiae. A yeast-2-
hybrid screen identified 15 novel protein-protein interactions not previously described for Sdm1,
including a yeast-2-hybrid interaction with Ace2. Furthermore, the uncharacterised gene,
YOL036W, a paralogue of Sdm1, was identified to interact with Sdm1, and the RAM proteins,
Mob2 and Cbk1, in a yeast-2-hybrid screen. We propose that Sdm1 and Yol036w may be
involved in ER to Golgi trafficking and thereby the localisation of glycosylated proteins, such as
Cts1, that are involved in cell separation.




                                                 103
P54C
APSES Proteins Play a Crucial Role for Nitrogen Utilization in Pathogenic Candida Species
Elena Lindemann, Christian Grumaz, Julia Küsel, Steffen Rupp and Kai Sohn
Molecular Biotechnology, Fraunhofer IGB, Nobelstr.12, Stuttgart 70569, GERMANY, Phone:
+49 (0)711 970 4145, FAX: +49 (0)711 970 4200, e-mail: lind@igb.fraunhofer.de, Web:
www.igb.fraunhofer.de

APSES proteins represent a class of transcription factors regulating cellular differentiation
processes in fungi. With the identification and functional characterisation of MOM1 (Modulator
of Morphogenesis 1) in Candida dubliniensis, which is highly homologous to the members of the
APSES family, we found that this gene is responsible for the regulation of morphogenetic
processes including filamentation as well as differentiation to chlamydospores. These results are
analogous to the morphologies found for efg1 deletion strains in Candida albicans. Strikingly,
despite a reduced ability to form filaments, only mom1 deletion strains, but not efg1 strains,
exhibit radially symmetric colony projections consisting of true hyphae on YCB-BSA agar plates,
indicating a divergent repressing function of MOM1 in C. dubliniensis, which has not been
established for EFG1 in C. albicans. Transcriptional profiling using MESSAGE as well as DNA
microarrays of wildtype and mom1 deletion strains under blastospore and hyphae inducing
conditions in Candida dubliniensis, not only revealed transcripts differentially expressed during
morphogenesis, but also downstream factors of MOM1 including Cd36_15230 (CdSTF2),
Cd36_43260 (CdECE1) and Cd36_13090 (CdRBE1). A significant downregulation for transcript
levels of glycolytic genes like Cd36_08010 (CdENO1) and Cd36_60670 (CdPGK1) in the mom1
deletion strains also shows that Mom1p (like Efg1p in Candida albicans) is implicated in the
expression of genes regulating carbon metabolism. Surprisingly, we found a not yet described
essential function of both proteins in the regulation of nitrogen utilization. Although mom1 and
efg1 were able to grow in media containing either ammonia or BSA as a sole nitrogen source, both
deletion strains in contrast to the respective WT were unable to grow under acidic conditions,
when both nitrogen sources were simultaneously present. These results indicate that Efg1p as well
as Mom1p might act as a molecular switch crucial for the adaptation to complex nitrogen
environments in different pathogenic Candida species.




                                              104
P55A
Distinct regions of Rac1 confer localization and functionality to this conserved G-protein
Hannah Hope, Danièle Stalder, Romain Vauchelles, Robert Arkowitz and Martine Bassilana
Institute of Developmental Biology and Cancer, CNRS UMR6543 - University of Nice-Sophia
Antipolis, Parc Valrose, Faculté des Sciences, Nice 06108, France, Phone: +33 (0)4 9207 6464,
FAX:         +33      (0)4     9207       6466,      e-mail:      mbassila@unice.fr,     Web:
http://www.unice.fr/isdbc/equipe/equipe.php?id=12

* The first three authors contributed equally to this work

In Candida albicans there are six Rho GTPases, some of which have been shown to have distinct
roles during polarized growth in response to different signals. For example the two highly
homologous Rho G-proteins, Rac1 and Cdc42, which are approximately 60% identical, are
required for filamentous growth in response to different stimuli (1). We analyzed the importance
of specific regions of Rac1 for its function and our results show that a 43 amino acid region,
unique to C. albicans Rac1, is required for invasive filamentous growth. Furthermore, Rac1
localizes to the plasma membrane and in certain conditions we observe accumulation of this G-
protein in the nucleus. Such a nuclear localization of Rac1 has been demonstrated in mammalian
cells (2) where, in particular, Rac1 nucleo-cytoplasmic shuttling is important for cell division (3).
We have determined the regions of Rac1 necessary for targeting to these distinct locations and
examined the dynamics of Rac1 both at the plasma membrane and in the nucleus. The carboxy-
terminus of Rac1 can target Cdc42 to the plasma membrane and conversely the carboxy-terminus
of Cdc42 can target Rac1 to the plasma membrane. Both of these chimeras are functional for
filamentous growth. The Rac1 carboxy-terminal CAAX sequence is necessary for its plasma
membrane localization, whereas the adjacent polybasic region is required for nuclear
accumulation. These regions are sufficient to target GFP to the plasma membrane and nucleus,
respectively. We have used fluorescence recovery after photobleaching (FRAP) methods to
investigate the dynamics of GFP-Rac1 at the plasma membrane and in the nucleus. Our results
indicate that Rac1 dynamics on the plasma membrane depends on nucleotide state of the G-
protein. Furthermore, upon photobleaching nuclear localized GFP-Rac1, a fluorescence recovery
is observed with a t1/2 of approximately 30 seconds. The contribution of different Rac1 regions to
its function and localization will be presented.

References
1. Bassilana M. and Arkowitz R. (2006) Eukaryot Cell, 5, 321.
2. Lanning C. et al. (2004) J. Biol. Chem., 279, 44197.
3. Michaelson D. et al. (2008) J. Cell. Biol., 181, 485.




                                                105
P56B
Characterisation of the first cell wall beta(1-3)glucan branching activity in Aspergillus
fumigatus.
Amandine Gastebois1, Thierry Fontaine1, Catherine Simenel1, Bernadette Coddeville2, Muriel
Delepierre1, Jean Paul Latge1 and Isabelle Mouyna1
1 Parasitology Mycology, Institut Pasteur, 25 rue du Docteur Roux, Paris 75015, France, Phone:
+33(0)1 45 68 82 25, FAX: +33(0)1 40 61 34 19, e-mail: agastebo@pasteur.fr, Web:
http://www.pasteur.fr/
2 Université des Sciences et Technologies de Lille, IFR 147, Villeneuve d'Ascq, France

The fungal cell wall is essential for fungal growth. Branched beta(1-3)glucan of A.fumigatus serve
as a skeleton on which other polysaccharides of the cell wall (chitin and galactomannan) are cross-
linked. Beta(1-3)glucan chains are extruded as a linear chain through the plasma membrane and
branched in the cell wall space.
We have identified a family of transglycosidase able to branch beta(1-3)glucans. These enzymes
have been isolated biochemically from cell wall autolysate of A.fumigatus.
The first one, AfBgt1p, was described earlier. This protein cleaves laminaribiose from the
reducing end of linear beta(1-3)glucan chain and transfer the remaining glucan to another beta(1-
3)glucan acceptor with a beta(1-6) linkage. This enzyme requires a free reducing end to display
it’s activity (Mouyna et al. 1998).
The second one is a GPI anchored protein which presents 21% identity with AfBgt1p but has a
different transglycosidase activity. This enzyme is able to cleave two residues from the reducing
end of beta(1-3)glucan chains and branch the remaining chain internally to another glucan chain
through a beta(1-6)linkage.
The recombinant protein has been produced in the yeast Pichia pastoris, this allowed a precise
characterisation of the enzymatic activity (optimum pH, optimum T°, Km). The gene named,
GBE1 , encoding this enzyme has been disrupted and the phenotype of the mutant is currently
analysed.
Mouyna I, et al, (1998),Microbiology 144, 3171.




                                               106
P57C
Role of the phosphoinositides in fungal polarized growth
Isabelle Guillas, Aurélia Vernay, Martine Bassilana and Robert Arkowitz
Institute of Developmental Biology and Cancer, CNRS UMR6543, Université Nice Sophia-
Antipolis Parc Valrose Cedex 02, Nice 06108, FRANCE, Phone: +33 (0)4 92 07 64 64, FAX: +33
(0)4        92        07       64        66,        e-mail:      vernay@unice.fr,    Web:
http://www.unice.fr/isdbc/equipe/equipe.php?id=12

*The first two authors contributed equally to this work

Candida albicans, similar to a range of human fungal pathogens, grows in different forms in
response to environmental cues. Its ability to switch from a yeast to a filamentous form in
response to different stimuli is important for pathogenicity. Membrane phospholipids such as
phosphoinositides, which are minor components of cellular membranes, have been shown to play
an important role in cell polarity in virtually all eukaryotes.
In Saccharomyces cerevisiae and Candida albicans, neither PI(3,4,5)P3 nor PI(4,5)P2-3-kinase
homologs have been found, raising the possibility that the PI(4,5)P2 fulfills some of PIP3’s
functions. Both organisms have a unique PI-4-Kinase (encoded by STT4) and PI(4)P-5-Kinase
(encoded by MSS4). MSS4 is an essential kinase required for the viability and actin cytoskeleton
organization in S. cerevisiae. Given the importance of PIP2 and PIP3 in external signal-mediated
polarized growth or movement, we examined whether PI(4,5)P2 is required for the yeast to
filamentous growth transition. Initially, we carried out a genetic screen in S. cerevisiae for mss4
mutants, from which we have isolated and characterized mutants specifically defective in invasive
growth that have in addition, reduced levels of PI(4,5)P2.
To investigate the role of PI(4,5)P2 in C. albicans filamentous growth we have generated strains
in which the level of the Stt4 or Mss4 phosphoinositide kinase can be manipulated in vivo , using
the Tetracycline repressible promoter system. The analyses of these C. albicans mutants, as well
as the aforementioned S. cerevisiae mutants, in response to different filamentous growth inducers
will be presented. In C. albicans we have focused on the response of these mutants to serum,
whereas in S. cerevisiae we have examined primarily invasive growth in response to reduced
glucose levels. Our results indicate that PI(4,5)P2 is important for the yeast to filamentous growth
switch and we are currently examining the requirement for this phospholipid’s function during
such morphological transitions.




                                                107
P58A
The Dbf2 kinase is essential for cytokinesis and correct mitotic spindle formation in Candida
albicans
Alberto González-Novo1, Leticia Labrador1, M. Evangelina Pablo-Hernando1, Jaime Correa-
Bordes2, Miguel Sánchez1, Javier Jiménez1 and Carlos Vázquez de Aldana1
1 Instituto Microbiologia Bioquimica, CSIC, Campus Unamuno, Salamanca 37007, Spain, Phone:
+34 923 252092, FAX: +34 923 224876, e-mail: cvazquez@usal.es
2 Dpto. Microbiología. Facultad de Ciencias. Universidad de Extremadura. Avda de Elvas s/n.
06071 Badajoz, Spain

Successful completion of cell cycle requires the coordination between different processes,
including DNA replication, chromosome segregation and cytokinesis. S. cerevisiae Mitosis Exit
Network (MEN) is a conserved pathway composed of several kinases (such as Cdc15 or Dbf2), a
phosphatase (Cdc14), a GTPase (Tem1) and its GEF (Lte1), that ensures timely coordination
between cytokinesis and exit from mitosis.
We have characterized the CaDBF2 gene, encoding a protein kinase of the NDR family in
Candida albicans, and demonstrated that, in contrast with its S. cerevisiae counterpart, this gene is
essential for cell viability. Conditional mutants were constructed by using the MET3 promoter to
analyze the phenotype of cells lacking this kinase. Absence of Dbf2 resulted in cells arrested as
large-budded pairs and actomyosin ring contraction failure. In addition to its role in cytokinesis,
CaDbf2 regulates mitotic spindle organization and nuclear segregation, since Dbf2-depleted cells
have abnormal microtubules and severe defects in nuclear migration to the daughter cell, which
results in a cell cycle block during mitosis. Taken together, these results imply that CaDbf2
performs several functions during exit from mitosis and cytokinesis. Consistent with a role in
spindle organization, Dbf2 localizes to the mitotic spindle during anaphase, and it physically
interacts with tubulin as indicated by immunoprecipitation experiments. Finally, CaDBF2
depletion also resulted in impaired true hyphal growth.




                                                108
P59B
Role and localization of Rho G-proteins in Candida albicans
Peter Follette, Olivier Pierre, Damian Bednarzcyk, Robert Arkowitz and Martine Bassilana
Institute of Developmental Biology and Cancer, CNRS UMR6543 - University of Nice-Sophia
Antipolis, Parc Valrose, Faculté des Sciences, Nice 06108, France, Phone: +33 (0)4 92 076425,
FAX:         +33       (0)4       92     076466,     e-mail:     arkowitz@unice.fr,      Web:
http://www.unice.fr/isdbc/equipe/equipe.php?id=12

Small Rho G proteins such as Rho1 and Cdc42 are key regulators of the actin cytoskeleton. In
Saccharomyces cerevisiae, Rho1 is required for viability and plays a critical role in cell wall
integrity via beta-1,3-glucan synthase, protein kinase C (Pkc1) and the actin cytoskeleton. In
Candida albicans Rho1, which has greater than 80% sequence identity with its S. cerevisiae and
human counterparts, was shown to be essential (1). Using mutants in which the sole copy of
RHO1 is under the control of the repressible Tetracycline promoter, we further investigated the
role of Rho1 in this organism. When RHO1 expression is repressed, mutant cells are unable to
grow and over-expression of a mutated form of Rho1, mimicking the GTP bound form, does not
restore viability. Congo Red inhibits growth in these non-repressed rho1delta/PTEToffRHO1
cells, confirming that Rho1 is necessary for cell wall integrity, yet sorbitol does not restore growth
of this mutant in the presence of such a cell wall perturbant. rho1delta/PTEToffRHO1 cells are
defective in filamentous growth in embedded media and on solid media containing serum, but
filament in liquid media containing serum, indicating that Rho1 is also required for filamentous
invasive growth. As both Rho1 and Cdc42 are critical for viability and filamentous growth, we set
out to examine the spatio-temporal regulation of these proteins, under different growth conditions.
Previous studies in S. cerevisiae have identified defined GTPase-binding domains (GBD) that
specifically bind an activated G-protein: the Pkc1 Rho Interaction Domain (RID) has been used to
localize activated Rho1 (2), while Cdc42/Rac-Interactive Binding (CRIB) domain from Gic2 was
used to localize activated Cdc42 (3). To determine the localization of activated Rho1 and Cdc42 in
C. albicans we used these two GBDs, respectively. To visualize activated Rho1, a fusion of the
RID from C. albicans Pkc1 with GFP was used whereas, because Gic2 is absent from the C.
albicans genome, we used the S. cerevisiae Gic2 CRIB domain fused to GFP to visualize
activated Cdc42. The localization of these sensors in wild-type, rho1 and cdc42 mutants exposed
to various stimuli will be presented.

References
1. Smith S. et al. (2002), FEMS Yeast Res., 2, 103.
2. Bar E. et al. (2003), J. Biol. Chem. 278, 2179.
3. Tong Z. et al. (2007), J. Cell. Biol. 179, 1375.




                                                 109
P60C
Transcriptional control of carbon metabolism in Candida albicans
Melissa Ramirez and Michael Lorenz
Microbiology and Molecular Genetics, The University of Texas Health Science Center, 6431
Fannin, Houston TX 77030, United States, Phone: +1 (713) 500-7422, FAX: +1 (713) 500-5499,
e-mail: Michael.Lorenz@uth.tmc.edu, Web: http://www.lorenzlab.org

Phagocytes of the innate immune system are a key component of mammalian defenses against
fungal pathogens. We and others have shown that phagocytosis by macrophages induces extensive
transcriptional changes in C. albicans, including a metabolic transition from glycolytic to
gluconeogenic growth. These pathways (beta-oxidation, the glyoxylate cycle, and
gluconeogenesis) are required for full virulence in a mouse model of disseminated candidiasis,
indicating that some niches in the mammalian host are carbon-poor. We have shown that deletion
of genes in these highly conserved pathways, such as ICL1 and FOX2 have unexpected pleiotropic
phenotypes in C. albicans – notably that the fox2 mutant fails to utilize ethanol as a carbon source.
The Distel group has shown that this is at least partly due to defects in peroxisome function in this
mutant. To shed more light on the mechanisms governing alternative carbon metabolism in C.
albicans, we have identified transcriptional regulators of these pathways based on Saccharomyces
cerevisiae and Aspergillus nidulans. C. albicans contains homologs of the S. cerevisiae Cat8p and
Adr1p (FacB and AmdX in A. nidulans) transcription factors that control expression of glyoxylate
cycle, gluconeogenic, and ethanol utilization genes. Null mutants of these genes confer no
apparent phenotype in vitro, in contrast to the corresponding S. cerevisiae mutants (but similar to
A. nidulans). C. albicans lacks Oaf1p and Pip2p, transcription factors that control peroxisome
biogenesis and beta-oxidation genes in yeast. Instead, it contains a homolog (Ctf1p) of the A.
nidulans fatty acid catabolism regulators FarA and FarB. We have shown that CTF1 is required
for growth on oleic acids (as are FarA/FarB) and that FOX2 expression is dependent on CTF1.
Also, in vivo, the ctf1 mutant showed a mild attenuation in virulence, similar to the fox2 mutant.
ctf1 mutant strains did not, however, confer pleiotropic phenotypes. Thus, both phenotypic and
genotypic observations suggest that the regulatory network for alternative carbon metabolism in C.
albicans appears more similar to filamentous fungi than budding yeast. We are currently
identifying targets of these regulators so as to understand more completely the observed
pleiotropic phenotypes and the roles of these pathways in vivo.




                                                110
P61A
Sit1-mediated siderophore utilization in the growth and virulence of Candida glabrata
Tracy Nevitt and Dennis J. Thiele
Pharmacology and Cancer Biology, Duke University Medical Center, Research Drive, LSRC,
Durham NC 27710, USA, Phone: +1 919 613 8197, FAX: +1 919 668 4060, e-mail:
tracy.nevitt@duke.edu

Microbial iron (Fe) acquisition is a major determinant for infection and persistence within a host.
The essential requirement for iron is reflected not only by the multiple mechanisms employed by
microbes to promote its active uptake, but notably by host iron-withholding strategies that
contribute towards attenuated microbial growth. So as to circumvent this, bacterial and fungal
pathogens express cell surface and secreted proteins and molecules capable of releasing iron from
host proteins and ligands.
Siderophores, small molecules with extremely high affinity for Fe3+, are ubiquitously utilized by
bacteria and fungi in the mobilization of extracellular Fe.
Candida glabrata, an increasingly relevant cause of fungemia in immunocompromised
individuals, does not synthesize siderophores, but is capable of utilizing xenosiderophores as an
Fe source. We identified SIT1 (SIderophore Transporter 1) in C. glabrata, whose translation
product exhibits significant similarity to S. cerevisiae Arn1 and Taf1 proteins. Expression analyses
show that, under Fe deficiency, SIT1 is highly induced and the protein localizes to the plasma
membrane, where the transporter mediates the utilization of hydroxamate-type siderophores. Our
results further show that the sit1 deleted mutant is compromised for growth under conditions
where siderophore substrates are the sole Fe source.
Given the essential nature of Fe, we hypothesized that the sit1 mutant may display attenuated
growth given the Fe-limiting environment of the mammalian host and the existence of a diverse
siderophore-producing microbiota. To address this, we challenged C. glabrata to the microbicidal
environment of the macrophage phagosome. Upon phagocytosis, macrophages actively deplete the
phagosome of Fe whilst concomitantly generating reactive oxygen species (ROS). Given the
critical role of Fe-binding proteins, such as catalase, in ROS detoxification we predicted that the
sit1-deleted strain would be more susceptible to macrophage killing than the wild type in the
presence of siderophores. Our results show that both the wild type and SIT1-reconstituted strains
show enhanced survival to a macrophage challenge when compared to the sit1 mutant strain.
Studies are now underway to extend these studies to a mouse model of C. glabrata candidiasis.
As a fungal-specific protein, the Sit1 transporter stands as an attractive target for the development
of novel antifungal therapeutics.




                                                111
P62B
The role of microtubules in hyphal growth in the human fungal pathogen Candida albicans
Laura Jones and Peter Sudbery
Molecular Biology and Biotecnology, University of Sheffield, Western Bank, Sheffield S10 2TN,
England,      Phone:      +44(0)1142222748,        FAX:       +44(0)1142222748,        e-mail:
mbp05laj@sheffield.ac.uk, Web: www.shef.ac.uk

The polarisation of growth to C.albicans hyphal tips requires the guidance of secretory vesicles
and other molecular cargo to the site of active growth. Both the actin and microtubular
cytoskeletons are thought to play a role in this localisation, although there are conflicting reports
regarding the importance of microtubules. When hyphal cells are treated with an actin-cable
disrupting chemical, swelling occurs at the tip and is concurrent with the loss in localisation of
polarity components from the Spitzenkorper. One study has shown that treatment of hyphae with
chemicals which disrupt tubulin, has no effect on morphology, instead growth appears to slow
down and eventually cease. However, other studies have reported no effect. This has left the role
of microtubules in C.albicans hyphae growth uncertain. We have shown that the C.albicans
homologue of N.crassa Kinesin-1p microtubule motor protein, CaKip4p, shows severe hyphal
specific growth defects when deleted. The mutant shows no defects in the expression of hyphal
specific genes, evidence the mutant is defective in the machinery that drives the formation of
hyphal germ tubes. Localisation of Kip4p-YFP to long cable like structures within the germ tube,
and time-lapse microscopy images of small structures moving along the cables, are consistent with
motor protein models in which kinesins “walk” along microtubules. Additionally, interaction
studies have provided further evidence that Kip4p is a true kinesin. This evidence suggests that
microtubules do in fact play an important role.
To further characterise the growing tips of hyphae, FRAP microscopy was used to investigate the
dynamic properties of the three tip structures; the polarisome, the exocyst and the Spitzenkorper.
The first two structures are found in all three morphological forms, the Spitzenkorper is unique to
hyphae. Firstly, two polarisome proteins showed the slowest recovery pattern. The middle group
contains members of the exocyst, the Myo2p regulatory protein Mlc1p, with the secretory vesicle
protein Sec2p showing marginally faster recovery. Interestingly, Sec4p, for which Sec2p is the
GEF, showed the fastest recovery rate, clearly distinct from the rest of the sample. This is an
intriguing finding, as based on data from S. cerevisiae, Sec2p recruits Sec4p to the vesicles, with
Sec4p being dependent on Sec2p for activation and localisation. These results suggest the model
from S.cerevisiae does not apply to C.albicans hyphae.




                                                112
P63C
CUG ambiguity in C. albicans is remodelled by environmental factors
João Simões, Ana Rita Bezerra and Manuel Santos
Biologia, Universidade de Aveiro, campus universitário de santiago, Aveiro 3810-193, Portugal,
Phone: +351 234 372 587, FAX: +351 234 370 350, e-mail: joaosimoes@ua.pt, Web:
http://www.ua.pt/ii/rnomics

The ascomycete Candida albicans is a normal resident of the gastrointestinal tract of humans and
other warm-blooded animals. This yeast is a successful commensal and a pathogen that occurs in a
broad range of body sites. It also has high capacity to survive and proliferate in environments with
drastic changes in oxygen, carbon dioxide, pH, osmolarity, nutrients and temperature. This
opportunistic pathogen and other Candida spp. have a unique genetic code due to change of
identity of CUG codon from leucine to serine. Previous studies showed that 3.0% of leucine and
97.0% of serine are incorporated at CUG codons in vivo under standard growth conditions and
that it increases under stress up to 5.0%. This suggests that CUG ambiguity is remodeled under
stress. In order to better understand this unique biological phenomenon we have developed a
fluorescent reporter system based on GFP to quantify leucine insertion at CUG positions in vivo.
The system is based on the plasmid pACT1-GFP, which contains the codon-optimized yeast
enhanced green fluorescent protein (yEGFP), where serine-201 encoded by the codon TTA was
mutated to the ambiguous CTG and also to TCT (serine) codon (negative control). C. albicans
cells expressing these recombinant GFPs were grown at 25ºC, 30ºC, 37ºC, 40ºC and 42ºC and in
other physiological conditions. GFP fluorescence was monitored using an epifluorescence
microscope and quantified using ImageJ software. The data shows increased CUG ambiguity with
increasing temperature, suggesting that misincorporation of leucine into the C. albicans proteome
increases during infection.

Acknowledgements: JS is supported by the Portuguese Foundation for Science and Technology
through the PhD grant REF: SFRH/BD/39146/2007.




                                                113
P64A
The metabolic response of Candida glabrata to phagocytosis
Andreas Roetzer, Nina Gratz, Pavel Kovarik and Christoph Schüller
Department of Biochemistry, University of Vienna, MFPL, Dr.Bohrgasse 9/5, Vienna A-1030,
Austria, Phone: +43 1 4277 52815, FAX: +43 1 4277 9528, e-mail:
christoph.schueller@univie.ac.at, Web: http://www.mfpl.ac.at/index.php?cid=81

Macrophages erase cells of the fungal pathogen Candida glabrata after phagocytosis. For the
fungal pathogen the phagosome is a unique hostile environment. To assess the response of C.
glabrata cells to phagocytosis we employed fluorescent protein fusions to catalase CgCta1,
CgYap1, and CgMig1 as in vivo reporters. C. glabrata catalase levels are regulated by oxidative
stress and glucose deprivation. During oxidative stress GFP-CgCta1 increases rapidly in the
cytoplasm, whereas growth with ethanol or oleic acid as carbon source accumulates it in
peroxisomal structures. These stain with a peroxisomal YFP-SKL reporter and depend on CgPex3
suggesting them to be bona fide peroxisomes. We find that immediately after phagocytosis C.
glabrata cells experience glucose starvation and transient oxidative stress. Nuclear accumulation
of GFP-CgYap1 and cytosolic accumulation of CgMig1-CFP fluorescence report these conditions
in engulfed cells. The response to phagocytosis further proceeds with transient appearance of
peroxisomes indicating a shift of metabolism. Most engulfed cells are able to divide after an initial
lag phase while the loss of expression of GFP-CgCTA1 predicts upcoming cell death. Our results
support that oxidative stress is a minimal burden for C. glabrata within macrophages whereas
carbon resources are a major limiting factor.




                                                114
P65B
Cell density regulation of growth, GXM release and melanization in Cryptococcus
neoformans
Patrícia Albuquerque de Andrade, André Moraes Nicola and Arturo Casadevall
Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue
F411, Bronx NY 10461, United States of America, Phone: 1-718-430-3766, FAX: 1-718-430-
8701, e-mail: pandrade@aecom.yu.edu, Web: http://www.aecom.yu.edu

Quorum sensing (QS) is a cell density dependent mechanism of communication between
microorganisms, mediated by quorum sensing molecules (QSMs) that are accumulated during cell
growth. When the QSMs reach a certain threshold concentration, they induce the entire population
to cooperate in behaviors such as bioluminescence, antibiotic production, sporulation, biofilm
formation and expression of virulence traits. Studied mostly in bacteria, eukaryotic QS was
unknown until the recent discovery of farnesol and tyrosol as QSMs controlling filamentation and
biofilm formation in Candida albicans. Due to the critical role described for QS in the regulation
of virulence in other pathogenic microorganisms, we have investigated its presence in
Cryptococcus neoformans. This encapsulated yeast is the etiologic agent of cryptococcosis, a life-
threatening systemic mycosis that predominantly affects immunocompromised people. To look for
QS activity, we tested the effect of C. neoformans conditioned medium (CM) in different
situations related to pathogenicity. CM had no effect in filamentation or mating. However, we
found significant dose-dependent effects of CM in cell growth, glucuronoxylomannan (GXM)
release and melanization, three of the most important virulence attributes of this organism.
Addition of conditioned medium to fresh C. neoformans cultures at low cell density resulted in a
dose-dependent decrease of the lag phase and faster replication. CM from all four serotypes of C.
neoformans and C. gattii induced this effect in growth, and all four serotypes responded with
faster growth as well. The effect was independent on the temperature in which the cultures were
incubated. We collected the supernatant of all the C. neoformans cultures used in the growth assay
and measured the GXM concentration by capture ELISA. Addition of CM resulted in a dose-
dependent increase in extracellular GXM for all cultures containing CM. Addition of similar
concentrations of CM into solid media containing L-DOPA, a melanin precursor, demonstrated a
significant decrease in the time needed for fungal melanization when compared to cultures in pure
L-DOPA medium. These results support the existence of QS regulation of multiple C. neoformans
virulence attributes. We are currently isolating and characterizing the QSM responsible for this
communication, as well as the genes involved in it.




                                               115
P66C
Identification of the Candida albicans Cap1p regulon
Sadri Znaidi1, Katherine S. Barker2, Sandra Weber1, Anne-Marie Alarco3, Teresa T. Liu2,
Geneviève Boucher1, P. David Rogers2 and Martine Raymond1
1 Yeast Molecular Biology, Institute for Research in Immunology and Cancer, 2950, chemin de
Polytechnique, Montreal QC H3T 1J4, CANADA, Phone: +15143436111 ext. 0670, FAX:
+15143437383, e-mail: sadri.znaidi@umontreal.ca, Web: www.iric.ca
2 Departments of Clinical Pharmacy, Pharmaceutical Sciences, Molecular Sciences, and
Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee, 38163, USA
3 Génome Québec, Montréal, Quebec, Canada H3B 1S6

Cap1p, a transcription factor of the basic region-leucine zipper family, controls the oxidative stress
response in Candida albicans and protects C. albicans against reactive oxygen species generated
upon exposure to neutrophils during the course of the immune response in the host. It was shown
that alteration of the C-terminal cysteine-rich domain (CRD) of Cap1p results in nuclear retention
and constitutive transcriptional activation, a mechanism reminiscent of the S. cerevisiae ortholog
Yap1p. To further characterize the function of Cap1p in C. albicans, we used genome-wide
location profiling (ChIP-on-chip), allowing the identification of Cap1p-transcriptional targets in
vivo. A triple-hemagglutinin epitope was introduced at the C-terminus of wild-type Cap1p
(Cap1p-HA) or hyperactive Cap1p with a mutated CRD (Cap1p-CSE-HA). Location profiling
using an oligonucleotide tiling DNA microarray identified 89 targets that were bound by Cap1p-
HA or Cap1p-CSE-HA (binding ratio > 2-fold, P < 0.01). Strikingly, Cap1p binding was not only
detected at the promoter region of its target genes but also at their 3'-end and within their open-
reading frame, suggesting that Cap1p may associate with the transcriptional or the chromatin
remodelling machinery to exert its activity. Cap1p binding was also enriched at “gene deserts”
suggesting that Cap1p may regulate small non-coding transcripts or yet unidentified genes.
Bioinformatic analyses suggested that Cap1p binds to the DNA motif 5'- MTKASTMA, which
includes the previously characterized Yap1-response element TTA(C/G)TAA. Overrepresented
functional groups of Cap1p targets (P < 0.02) included 11 genes involved in response to oxidative
stress (CAP1, GLR1, TRX1, others), 13 genes involved in response to drug (PDR16, MDR1,
FLU1, others), 5 genes involved in hyphal cell wall function (PDC11, SSA2, orf19.251, others), 4
genes involved in phospholipid transport (PDR16, GIT1, RTA2 and orf19.932) and 3 genes
involved in regulation of nitrogen utilization (orf19.2693, orf19.3121 and GST3). Transcriptome
analyses showed that increased expression of most Cap1p targets accompanies Cap1p binding at
these targets, indicating that Cap1p is a transcriptional activator. Our results suggest that, in
addition to protecting the cells against oxidative stress, Cap1p has other important functions
including drug resistance and the regulation of nitrogen utilization.




                                                 116
P67A
Candida albicans general amino-acid permease transporters (CaGaps)
Lucie Kraidlova1, Helene Tournu2, Patrick van Dijck2 and Hana Sychrova1
1 Department of Membrane Transport, Institute of Physiology, Videnska 1083, Prague CR 142 20,
Czech Republic, Phone: +420 777 856 658, FAX: +420 296 442 194, e-mail:
kraidlova@biomed.cas.cz
2 Dept. Molecular Microbiology, VIB, Lab. Molecular Cell Biology, K.U. Leuven, Institute of
Botany and Microbiology, Kasteelpark Arenberg 31, B-3001 Leuven, Belgium

Candida albicans can proliferate in many different niches within the host. Therefore it must be
able to sense its environment very well in order to express only those genes required to proliferate
in that area. There is some evidence that sensing and uptake of amino acids is very important for
Candida albicans cells for growth in the host as well as for virulence. In Saccharomyces
cerevisiae, it was recently found that the general amino-acid permease Gap1 is not only required
for amino-acid transport, but also for sensing the presence of amino acids and thereby activating
signal transduction pathways that induce many intracellular changes. In Candida albicans, there is
a whole family (6 members) of ScGap1 orthologues. Our aim is to elucidate the role of individual
CaGap permeases in amino-acid uptake and sensing, in cell morphology, virulence and
pathogenicity. For this, we have employed two strategies: 1) deletion of GAP alleles in the
Candida albicans SN87 strain to construct first single deletion strains, and then double and triple
deletion mutants; and 2) heterologous expression of individual CaGAP genes in Saccharomyces
cerevisiae mutant strains lacking their own amino-acid permeases. The phenotypes of constructed
strains have been analyzed. For Candida albicans mutants, the growth and morphology tests on
different sources of carbon and/or nitrogen, as well as the study of expression regulation have
revealed different functions for individual CaGaps. This observation was confirmed in
Saccharomyces cerevisiae, where the substrate specifity of the products of individual CaGAP
genes have been estimated and the role of CaGAP2 in signaling confirmed.
This work was supported by the Czech grant MSMT LC 531, and by the Czech-Flemish bilateral
project 1-2006-06.




                                                117
P68B
Rim 8, an arrestine-like protein, as member of Rim101 pathway in Candida albicans
Jonathan Gomez-Raja and Dana Davis
Microbiology, University of Minnesota, 420 Delaware St, Minneapolis MN 55455, US, Phone: +1
612-624-7994, FAX: +1 (612) 626-0623, e-mail: gomez131@umn.edu

Candida albicans is a normal commensal of the human mucosa, but under some circumstances,
including immunosuppression, it can cause superficial or disseminated infections with severe
morbidity and mortality. The ability of C. albicans to cause disease, and presumably survive as a
commensal, depends on its ability to switch between the yeast and filamentous growth forms. The
Rim101 pathway controls the yeast-filament transition in response to extracellular pH and is
required for wild-type pathogenesis. The Rim101 pathway governs adaptation to neutral-alkaline
pH environments by activating the transcription factor Rim101. When C. albicans is in neutral-
alkaline environments, such as the blood stream, Rim101 is activated by proteolytic cleavage of
the 85 kD full length form to the 74 kD active form. Proteolytic activation of Rim101 requires a
number of upstream pathway members including Rim8, an arrestin-like protein. Although Rim8 is
required for Rim101 activation, previous studies demonstrated that RIM8 is transcriptionally
repressed at alkaline pH. Here, we have shown that Rim8 protein is modified and rapidly degraded
upon shift to neutral-alkaline pH and that Rim8 degradation is correlated with Rim101 processing.
Rim8 degradation, but not modification, requires Vps4 suggesting that Rim8 may be taken up into
multi-vesicular bodies and degraded in the vacuole. Support for this model comes form the fact
that at acidic pH, Rim8 is localized at the plasma membrane; at alkaline pH, Rim8 is localized to
intracellular sites. Finally, using co-immunoprecipitation assays, we have found that at alkaline
pH, Rim8 associates with Rim101 and an additional ubiquitin-modified protein. This suggests that
at alkaline pH, Rim8 is part of a multi-protein complex that promotes Rim101 processing.




                                              118
P69C
Understanding the role of the Candida albicans Yak1 kinase in the regulation hyphal growth
Audrey Nesseir1, Murielle Chauvel1, Dorothée Diogo1, Arnaud Firon1, Tristan Rossignol1, Sophie
Goyard1, Florian A.R. Kaffarnik2, Laura Selway3, Scott C. Peck4 and Christophe d’Enfert1
1 Fungal Biology and Pathogenicity, Institut Pasteur, 25 rue Docteur Roux, Paris 75015,
FRANCE, Phone: +33 (0) 1 4061 3126, FAX: +33 (0) 1 4568 8938, e-mail:
audrey.nesseir@pasteur.fr
2 The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich, NR4 7UH, UK
3 School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen, AB25 2ZD, Scotland, UK
4 Division of Biochemistry, 271H Bond Life Sciences Center, University of Missouri-Columbia,
Columbia, MO 65211, USA

C. albicans virulence is directly linked to its ability to switch between yeast and hyphal forms.
This conversion is also essential to form biofilms, a structure highly resistant to antifungals. Signal
transduction pathways regulating the yeast-to-hypha transition in response to environmental cues,
like physiological pH and temperature, or presence of serum, have been characterized. The
interplay between these pathways is not fully understood. Recently, we have uncovered a role for
the C. albicans Yak1 kinase in the regulation of the yeast-to-hypha transition and in the
maintenance of hyphal growth. Genetic and transcriptional studies have suggested that the Yak1
kinase may modulate the repression exerted by the Tup1 general repressor on hypha-induced
genes. As Yak1 harbors four putative phosphorylation sites for the cAMP-dependent protein
kinase (PKA), it may provide a link between positive regulation of the yeast-to-hypha transition
mediated by the cAMP signaling pathway and negative regulation mediated by the Tup1-Nrg1 and
Tup1-Rfg1 complexes.
In order to deepen our understanding of the role of Yak1 in morphogenesis, we are using several
approaches. First, six putative phosphorylation sites in Yak1 have been mutated in order to test
whether phosphorylation of Yak1 is necessary for its function. Second, we are using an over-
expression approach in order to identify genes acting upstream and downstream from Yak1 : we
have used the Gateway™ technology to establish a partial ORFeome of C. albicans. 416 ORFs
encoding 191 transcription factors, 86 protein kinases, 36 protein phosphatases and 103 proteins
with a putative role in signaling have been cloned in a Gateway donor vector and subsequently
transferred into C. albicans expression vectors allowing PKC1p-driven over-expression of TAP-
tagged proteins or TETp-driven overexpression of untagged proteins. A screen in a wild-type
strain has identified 16 genes whose over-expression triggers pseudo-hyphal or hyphal growth
under conditions normally conducive to yeast growth. These include genes already known for
their role in morphogenesis (eg RFG1 and CCN1) and genes whose contribution to morphogenesis
had not yet been uncovered. Current experiments aimed at evaluating whether the morphogenetic
phenotype associated to over-expression of these genes requires the function of Yak1 and other
regulators of morphogenesis and at identifying suppressors of the yak1/yak1 mutation under
hypha-inducing conditions will be reported.




                                                 119
P70A
Minimal requirements for DNA binding and transcriptional activity of C. albicans
transcription factor Tec1p
Klaus Schröppel, Walid Abu Rayyan, Anurag Singh and Miriam Sehnal
Medical Microbiology and Hygiene, University of Tübingen, Elfriede-Aulhorn-Str. 6, Tübingen
72076, Germany, Phone: +49 (0)7071 29 82358, FAX: +49 (0)7071 29 5440, e-mail:
klaus.schroeppel@med.uni-tuebingen.de

Hyphal growth and the transcriptional regulation of adaptation to the host environment are key
issues during the pathogenesis of the most frequent human fugal pathogen C. albicans. Tec1p, a
member of the TEA transcription factor family, has previously been shown to be involved in the
signaling events that lead to hyphal formation and gene regulation. A tec1/tec1 deletion mutant
does no longer form hyphae in vitro and is avirulent in vivo. Earlier studies showed that some
TEA transcription factors recognize and bind to the conserved DNA sequence 5’-CATTCY-3’,
which has been termed TEA consensus sequence, TCS. It can be detected in several promoters of
hyphae regulated genes of C. albicans. However, other studies suggested that Tec1p does not just
bind to TCS DNA, but interaction with additional coactivators is of similar importance for Tec1p
to exert its regulatory function. Since we are interested in the molecular mechanisms underlying
the Tec1p-dependant morphogenetic development and virulence gene regulation, we characterised
the interaction of TCS and Tec1p. The role of the C-terminus of Tec1p in the protein-DNA-
complex formation was addressed by application of C-terminally truncated rTec1p proteins in
DNA-binding assays. Modifications of the TCS sequence were analysed for their effects on
rTec1p-DNA interaction. rTec1p binds to a degenerate TCS DNA motif, which highlights the
need for an accurate evaluation of Tec1p responsive promoter elements; mere elimination of a
single type of TCS sequence based on computational decisions may yield incomplete data due to
additional types of funtionally active TCS, which serve as Tec1p-binding sites.




                                              120
P71B
Candidemia In Hospitalized Patients In Almaty
Bahyt Menshik and Almas Begdullayev
Pharmacology and biochemistry, Scientific center for drug research "KazBioMed", 15 Toraigyrov
st., ap. 22, Almaty 050043, Kazakhstan, Phone: +77051900585, FAX: +77272206467, e-mail:
space-rover@mail.ru

During prospective study carried out in 2006-2008 risk factors, spectrum of pathogens, treatment
options and mortality in 75 episodes of candidemia in 75 patients from 16 Almaty’s hospitals have
been analyzed. Majority (84%) of patients with candidemia were hospitalized in intensive care
units (ICU), 13% – in therapeutic wards and 3% – in surgical one. The main reasons for admission
in ICU were operations (36%), neoplasms (33%) and traumas (13%). Etiologic agents of
candidemia were Candida parapsilosis (32%), C.albicans (28%), C.tropicalis (10%), C.glabrata
(8%), &#1057;.guilliermondii (2%), C.famata (2%), C.zeylanoides (2%), C.rugosa (2%),
&#1057;.krusei (2%), C.lusitaniae (2%), Candida sp. (10%). Two and more species of Candida
spp. has been isolated from blood in 9% of patients. Localized lesions in different organs have
been found in 38% of patients. Antifungal therapy was administered to 60% of patients;
intravenous catheters were removed in 68%. Total mortality within 30 days after diagnosis of
candidemia was 49%. Mortality in patients who received antifungal therapy was 37%, in patients
without specific therapy – 72%. Mortality after removal of intravenous catheter was 35%, without
removal – 92%.




                                              121
P72C
Role of Ndt80p in sterol metabolism regulation and azole resistance in Candida albicans
Adnane Sellam and Andre Nantel
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount,
Montreal QC H4P 2R2, Canada, Phone: +1 (514) 496-6370, FAX: +1 (514) 496-9127, e-mail:
andre.nantel@nrc-cnrc.gc.ca

The Ndt80p transcription factor modulates azole tolerance in Candida albicans by controlling the
expression of the gene for the drug efflux pump Cdr1p. To date, the contribution of this
transcriptional modulator to drug tolerance are not yet well understood. Here we investigate the
role of Ndt80p in mediating fluconazole tolerance by determining its genome-wide occupancy
using Chromatin Immuno-Precipitation coupled to a high-density tiling array. Ndt80p was found
to bind a large number of gene promoters of diverse biological functions. Gene ontology analysis
of these Ndt80p targets revealed a significant enrichment in gene products related to cell wall,
carbohydrate metabolism, stress responses, hyphal development and cell cycle. Notably, a
significant enrichment was found for genes related to multidrug transport (P = 2.09e-04) including
drug transporters (CDR1,2,4 and MDR1). Ndt80p was found on the promoters of ergosterol
biosynthesis genes including the azole target Erg11p. Additionally, expression profiling was used
to identify fluconazole responsive-genes that require Ndt80p for their proper expression. We
found that Ndt80p is crucial for the expression of numerous fluconazole-responsive genes,
especially genes involved in ergosterol metabolism. Therefore, by combining genome-wide
location and transcriptional profiling, we have characterized the Ndt80p fluconazole-dependent
regulon and demonstrated the key role of this global transcriptional regulator in modulating sterol
metabolism and drug resistance in C. albicans.




                                               122
P73A
Identification of mutant variants of multidrug transporter CaCdr1p of Candida albicans
which display substrate specificity and uncoupling between drug transport and ATP
catalysis.
Nidhi Puri and Rajendra Prasad
Membrane Biology Laboratory (MBL), Jawaharlal Nehru University (JNU), School of Life
Sciences (SLS), New Delhi 110067, INDIA, Phone: +91-11-26704509,+91-9810974589, FAX: +
91-11-26741081, e-mail: npuri79@gmail.com

In view of the importance of Candida Drug Resistance Protein (Cdr1p) of pathogenic Candida
albicans in azole resistance, we have characterized its ability to efflux variety of substrates by
subjecting its entire transmembrane segment (TMS) 5 to site directed mutagenesis. All the mutant
variants of putative 21 amino acids of TMS5 and native CaCdr1p were overexpressed as a GFP-
tagged protein in a heterologous host Saccharomyces cerevisiae. Based on the drug susceptibility
pattern, the mutant variants could be grouped into two categories. The variants belonging to first
category were susceptible to all the tested drugs, as compared to those belonging to second
category which remained resistant to selective drugs. Interestingly, the mutant variants showed
uncoupling between ATP hydrolysis and drug efflux. The ATPase activity of all the mutant
variants remained unaffected; however, their ability to efflux representative drug substrates was
abrogated. Based on the competition experiments, we could identify TMS5 residues which are
specific to interact with select drugs. Our results provide first evidence of set of mutant variants of
CaCdr1p which display futile ATPase activity uncoupled to drug efflux. TMS 5 of Cdr1p thus not
only imparts substrate specificity but appears to act as a communication helix between ATP
catalysis and drug transport.




                                                 123
P74B
Curcumin Modulates Efflux Mediated By Yeast ABC Multidrug Transporters And Is
Synergistic To Antifungals
Monika Sharma1, Raman Manoharlal1, Suneet Shukla2, Suresh Ambudkar2 and Rajendra Prasad1
1 Membrane Biology Laboratory (MBL), Jawaharlal Nehru University (JNU), School of Life
Sciences (SLS), New Delhi 110067, INDIA, Phone: +91-11-26704509,+91-9958411024, FAX: +
91-11-26741081, e-mail: monikabiotech@gmail.com
2 Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, MD 20892, USA

Curcumin (CUR), a natural product of turmeric, from rhizomes of Curcuma longa are known
reversal agents of drug resistance phenotype in cancer cells over-expressing ATP-binding cassette
(ABC) transporters viz. ABCB1,ABCG2 and ABCC1. In the present study, we evaluated whether
CUR, could also modulate multidrug transporters of yeasts that belong to either the (ABC) or the
major facilitator superfamily (MFS). The effect of CUR on multidrug transporter proteins was
demonstrated by examining Rhodamine 6G (R6G) efflux in Saccharomyces cerevisiae cells
overexpressing ABC transporters CaCdr1p and CaCdr2p and MFS CaMdr1p of Candida albicans
and ScPdr5p of S. cerevisiae. CUR decreased the extra cellular concentration of R6G in ABC
transporter expressing cells while had no effect on R6G efflux mediated through MFS transporter
CaMdr1p. CUR competitively inhibited R6G efflux and the photo labeling of CaCdr1p by a drug
substrate prazosin analog [125I]-iodoarylazidoprazosin (IC50, 14.2 µM). Notably, the mutant
variants of CaCdr1p which displayed abrogated efflux of R6G also showed reduced modulation
by CUR. Drug susceptibility testing of ABC protein expressing cells by spot assays revealed that
CUR when combined was selectively synergistic with drug substrates such as R6G, ketoconazole,
itraconazole, and miconazole but not with fluconazole, anisomycin, cycloheximide, FK520. Taken
together, our results provide the first evidence that CUR modulates ABC multidrug transporters
and could be exploited in combination with conventional antifungal drugs to reverse MDR in
Candida cells.




                                              124
P75C
Transcriptional activation and Post-transcriptional regulation involving differential RNA-
protein interaction(s) and poly(A) tail length of CDR1 mRNA are the major determinants of
azole resistance in clinical isolates of Candida albicans
Raman Manoharlal1, Jyotsna Gorantala2, Monika Sharma1 and Rajendra Prasad1
1 Membrane Biology Laboratory (MBL), Jawaharlal Nehru University (JNU), School of Life
Sciences (SLS), New Delhi 110067, India, Phone: +91-11-26704509,+91-9871738536, FAX: +
91-11-26741081, e-mail: ramanbiotech@gmail.com
2 School of Biotechnology,Jawaharlal Nehru University, New Delhi -110067, India

Many azole-resistant (AR) clinical isolates of Candida albicans display an increased expression of
the drug transporters CDR1/CDR2. We evaluated the molecular mechanisms that contribute to the
maintenance of constitutively high CDR1 transcript levels in two matched pairs of azole-
susceptible (AS) and azole-resistant (AR) clinical isolates of C. albicans. To address this, we use
reporter constructs of GFP and lacZ fused either to the CDR1 promoter (PCDR1-GFP/lacZ;
transcriptional fusion) or to the CDR1 ORF (PCDR1-CDR1-GFP/lacZ; translational fusion)
integrated at the native CDR1 locus. It was observed that expression of the two reporter genes as
transcriptional fusion in the AR isolates is higher than in matched AS isolates. However, the fold
difference in the reporter activity between the AS and the AR isolates is even higher for the
translational fusions, indicating that the sequences within the CDR1 coding region also contribute
to its increased expression in AR isolates. Further analysis of these observations by transcription
run-on and thiolutin chase assays demonstrated a ~5-7 and 3-fold difference in the transcription
initiation rates and half-life of CDR1 mRNA in the AR as compared to their respective matched
AS isolates. Our results demonstrate that both increased CDR1 transcription and enhanced CDR1
mRNA stability contribute to the over expression of CDR1 in azole-resistant C. albicans isolates.
Swapping of heterologous and chimeric lacZ-CDR1 3'UTR transcriptional reporter fusion did not
alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the
CDR1 3'UTR itself are not sufficient to confer the observed differential mRNA decay. An RNA-
Electrophoretic Mobility Shift Assay showed reduced binding of trans-regulatory factor(s) in AR
isolates. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was ~35% to 50%
hyperadenylated compared with AS isolates. Our study provided first evidence that differential
RNA-protein interaction(s) and hyperadenylation of CDR1 3'UTR rather than cis-acting sequences
within the 3'UTR are important potential determinants of mRNA stability in AR isolates.




                                               125
P76A
In vitro investigation of the activity of miconazole against biofilms of various Candida
species
Davy Vandenbosch, Hans J. Nelis and Tom Coenye
Laboratory of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, Ghent 9000,
BELGIUM, Phone: +32 (0)9 264 80 93, FAX: +32 (0)9 264 81 95, e-mail:
davy.vandenbosch@UGent.be

Biofilms formed by Candida species consist of a dense network of cells, hyphae and
pseudohyphae embedded in an extracellular matrix. Biofilms are highly resistant against
antifungal agents and there is an increasing need for effective antifungals. Azoles have a
fungistatic effect based on the inhibition of the enzyme 14-alpha-demethylase in the ergosterol
biosynthesis. Previous research showed that miconazole (an imidazole) has also fungicidal activity
associated with the induction of ROS (reactive oxygen species). In the present study the fungicidal
activity of miconazole against in vitro grown Candida biofilms has been investigated.
Furthermore, the relationship with the production of ROS was examined.
Biofilms of ten Candida albicans strains and five other Candida species were grown for 24 h on
silicone disks. The effect of miconazole (5 mM) on these mature biofilms was investigated by
plating. The level of ROS induction in planktonic and sessile cells was determined using a
fluorometric assay with DCFHDA (2’,7’-dichlorofluorescein diacetate). The MIC (minimal
inhibitory concentration) of miconazole was determined according to the EUCAST protocol. All
experiments were performed in the absence and presence of ascorbic acid (10 mM), a quencher of
ROS activity.
Miconazole showed a significant (p < 0.05) fungicidal effect against mature Candida biofilms.
Furthermore, miconazole strongly induced ROS production both in planktonic and sessile cells.
The addition of ascorbic acid to miconazole-treated planktonic Candida cells drastically reduced
ROS production for all strains. A simultaneous decrease in susceptibility to miconazole was
observed for most (10) strains. In contrast, the significant quenching of ROS after addition of
ascorbic acid to Candida biofilms did not lead to a reduction of the fungicidal activity of
miconazole.
In conclusion, the fungicidal activity of miconazole against Candida biofilms may be of
importance in the treatment of biofilm-related Candida infections. An increased ROS production
was observed during miconazole treatment, however this was not directly related to the fungicidal
activity of miconazole against Candida biofilms.




                                               126
P77B
PROTEOME analysis of the response of Aspergillus fumigatus to voriconazole and the role
of the cross-pathway control system in drug resistance
Nansalmaa Amarsaikhan, Olaf Kniemeyer and Zumrut Ogel
Biotechnology, Middle East technical University, ODTU, Ankara 06531, Turkey, Phone:
+905556411509, FAX: +903122102767, e-mail: nansa89@yahoo.com

Aspergillus fumigatus is the most important airborne fungal pathogen which can cause invasive
aspergillosis in immunocompromised individuals, such as transplantation patients. The diagnosis
of Aspergillus infections is difficult and often ambiguous. In addition, the number of available
antifungal compounds is rather limited. Aspergillosis is usually treated by the application of
antifungal compounds, in most cases by drugs of the azole group such as voriconazole,
posaconazole etc. Recently, there has been increasing evidence for antifungal drug resistance in
Aspergillus. For this reason, the research focus has shifted to investigating the key proteins
involved in drug resistance mechanisms. Commonly, it is known that development of antifungal
drug resistance is associated with the upregulation of general stress response pathways. Thus,
studies focusing on the transcriptional and proteomic profiles are of great importance to address
these general mechanisms.
Fungal CpcA is the functional orthologue of the yeast transcriptional activator protein Gcn4p. It is
a key protein in the regulation of the fungal amino acid biosynthesis which is vital to metabolism
with feeding substrates entering from various metabolic routes. Hence, it is a global regulatory
system modulating fungal amino acid biosynthesis as a whole and commonly referred to as the
cross-pathway control (cpc) or general control of amino acid biosynthesis. Apart from its role in
general amino acid control, its role in virulence of A. fumigatus has been revealed, supporting the
function of the fungal cross-pathway control of amino acid biosynthesis as a general stress
response system.
This study aims at confirming the central role of CpcA in stress response and assigning a
particular role of this protein in the antifungal drug resistance. In this study, we are planning to
study the change of the protein expression level of A.fumigatus in response to voriconazole, an
important azole group drug. Besides, we want to compare the response of wild type and cpcA
deleted strains of A.fumigatus in the presence of voriconazole. As a result of this study, we will be
able to compare the proteome data with transcriptome data released in 2006. Thus, we are
interested in comparing the proteomic profile of wild-type and delta cpcA strains exposed to
voriconazole.




                                                127
P78C
A nucleotide sugar transporter crucial for galactofuranosylation in Aspergillus fumigatus
Jakob Engel, Philipp S. Schmalhorst, Rita Gerardy-Schahn, Hans Bakker and Françoise H.
Routier
Cellular Chemistry, Medical School Hanover, Carl-Neuberg-Str. 1, Hannover D 30625, Germany,
Phone: +49 511 5323367, FAX: +49 511 5323956, e-mail: engel.jakob@mh-hannover.de, Web:
http://www.mh-hannover.de/

The human pathogenic fungus Aspergillus fumigatus is responsible for the severe and often fatal
disease Invasive Aspergillosis which occurs in immunocompromised patients. Among the most
effective antifungal agents launched so far are inhibitors of beta-1,3-glucan synthesis, an abundant
fungal cell wall polysaccharide. Another major cell wall component of A. fumigatus is
galactomannan, a polysaccharide composed of mannose and galactofuranose (Galf). The unusual
sugar Galf is present at the surface of many pathogenic organisms including bacteria, fungi and
parasites but absent from higher eukaryotes. We have recently demonstrated that the absence of
Galf decreases the virulence of A. fumigatus and increases its sensitivity to front line drugs [1].
The Galf biosynthetic pathways are thus attractive targets for adjunct therapy of Invasive
Aspergillosis.
The enzyme UDP-galactopyranose mutase which is responsible for the synthesis of the activated
nucleotide sugar UDP-Galf has recently been identified and localized to the cytosol [2]. Transport
of UDP-Galf into the Golgi lumen, where galactofuranosylation of N-glycans and glycolipids
takes place, is thus necessary. In the genome of A. fumigatus we identified 17 nucleotide sugar
transporter (NST) genes. Targeted gene deletion based on these candidates led to the identification
of glfB, encoding a UDP-Galf specific NST. This was first demonstrated in western blot analysis
using a cell wall extract of the DeltaglfB mutant, which did not react with the Galf specific
monoclonal antibody EB-A2. Moreover glycolipids and N-glycans, both galactofuranosylated in
the wild type fungus, were purified and analyzed by high performance thin layer chromatography
and capillary electrophoresis, respectively. These analyses revealed a complete Galf deficiency of
the DeltaglfB mutant, indicating also that in contrast to chitin and glucan, the biosynthesis of
galactomannan seems to take place along the secretory pathway.
The specificity of the NST was studied by an in vitro transport assay using Golgi vesicles isolated
from yeast overexpressing glfB. The Transporter was shown to be inactive towards a range of
UDP-sugars, including UDP-galactopyranose. In contrast, transport of UMP could be inhibited by
UDP-Galf, demonstrating that the transporter identified in this study is specific for UDP-Galf.




                                                128
P79A
Expression of virulence genes in Candida albicans biofilms grown in different biofilm model
systems
Heleen Nailis, Dieter Deforce, Hans Nelis and Tom Coenye
Lab of Pharmaceutical Microbiology, Ghent University, Harelbekestraat 72, Gent 9000,
BELGIUM, Phone: +32(0)92648141, FAX: +32(0)92648195, e-mail: tom.coenye@ugent.be,
Web: http://www.ugent.be/fw/en/research/pharmaceutical-analysis/pmicro

Candida albicans is a commensal of the human flora, but this fungus can also cause severe
superficial and systemic infections. The transition from commensal to pathogen is associated with
changes in the expression of genes encoding virulence factors. Virulence factors include secreted
aspartyl proteases (SAP), lipases (LIP), phospolipases (PLB) and agglutinin-like sequence (ALS)
proteins. These enzymes (Saps, Lips and Plbs) and adhesins (Als) probably play an important role
in the infection process. On biotic and abiotic surfaces, C. albicans can form biofilms, consisting
of a three-dimensional structure of yeast cells and filaments embedded in an extracellular matrix.
Biofilm formation also seems to be an important virulence factor since the majority of Candida
infections are biofilm-related. However, the expression of virulence genes in biofilms has not yet
been investigated and it is not known which molecular mechanisms are important for biofilm-
associated virulence.
The aim of the present study was to quantify the expression of genes belonging to the SAP, LIP,
PLB and ALS families in C. albicans biofilms grown in different model systems using RT-
quantitative PCR. Biofilms were grown in vitro on silicone disks in 24-well microtiter plates and
in a continuous flow system, the CDC reactor. Biofilms were also grown in an ex vivo model
using reconstituted human epithelial (RHE) cells.
Our data show that at each particular time point of biofilm growth in each model system, several
ALS genes are significantly upregulated in biofilms, compared to planktonic cells (p<0.05).
Furthermore, all LIP and PLB genes were highly overexpressed, but only in in vitro grown mature
biofilms. Some SAP genes were overexpressed (SAP1, SAP6, SAP7, SAP8 and SAP10) and
others were underexpressed (SAP3-5) in in vitro grown biofilms. In in vivo grown biofilms SAP4-
6 were upregulated and SAP2-3 downregulated.
Taken together, sessile cells exhibit a specific expression of SAP, LIP, PLB and ALS genes,
probably resulting in a biofilm-specific production of virulence factors. In addition, our data
highlight the importance of using multiple model systems in order to investigate the expression of
genes associated with virulence in C. albicans biofilms.




                                               129
P80B
Small molecule inhibitor of Candida albicans invasion specifically blocks hyphal elongation
Michael La Fleur, Chunfeng Xie and Kim Lewis
Antimicrobial Discovery Center, Northeastern University, 360 Huntington Avenue, Boston MA
02115, USA, Phone: 1-617-373-5013, FAX: 1-617-373-3724, e-mail: lafleur.m@neu.edu, Web:
http://www.northeastern.edu

Candida albicans biofilms are notoriously resistant to antimicrobials and cause important clinical
complications. We performed a high throughput screen in order to identify small molecule
miconazole potentiators active against C. albicans biofilms. Several potentiators were identified
and compound AC17 had excellent stability along with other desirable drug-like properties. AC17
was subsequently found to prevent invasion by Candida into solid media and appears to be a
specific inhibitor of hyphal elongation in liquid media. The inhibition of invasion by AC17
occurred at low concentrations (<10 µg/ml) and was consistent for all media and growth
conditions that were tested including Lee’s, Spider, RPMI and for cells embedded in YPS agar.
Interestingly, AC17 did not inhibit the growth of yeast cells cultured in YPD liquid medium and
did not prevent the yeast to hyphae transition, as has been reported for other small molecules.
AC17 may target a transcription factor required to maintain hyphal growth or elongation, such as
UME6. Future experiments will be aimed at testing whether AC17 has in vivo efficacy and
identifying its target.




                                               130
P81C
Investigations into the anti-C. albicans activity of a synthetic decapeptide with yeast killer
toxin like activity
Raymond Rowan, Gary Moran, Derek Sullivan, David Coleman and Luciano Polonelli
Oral Microbiology, Dublin Dental School & Hospital, Lincoln Place, Dublin ABC1234, Rep of
Ireland, Phone: 00353 85 7735908, FAX: 0035316711255, e-mail: raymond.rowan@dental.tcd.ie

The increasing incidence of fungal infections within immuno-compromised patients and the
emergence of isolates resistant to the currently used anti-fungals have prompted the search for the
next generation of antimycotics. Anti-fungal peptides have large potential for development as
novel therapeutic antimicrobial agents. Killer peptide (KP) is a synthetic decapeptide derived from
the sequence of a single-chain recombinant anti-idiotypic antibody raised against Pichia anomola
killer toxin. This peptide has been reported to exert activity both in vitro and in vivo in a model of
mucosal and systematic candidiasis. In addition to the potent anti-Candida albicans activity of KP,
its spectrum of activity includes activity against multidrug resistant Staphylococcus aureus,
Mycobacterium tuberculosis, Streptococcus pneumoniae and Enterococcus faecalis isolates.
Furthermore, KP also demonstrates potent anti-HIV and anti-influenza-1 activity. Surprisingly,
very little is known about the anti-microbial mechanism of action of this KP.

In order to investigate the molecular basis of how KP exerts its anti-candidal effects , we have
investigated the transcriptional response of C. albicans to KP using micro-arrays and RT-PCR.
Investigations have revealed an increase in the expression of the genes coding for chitin synthesis,
oligopeptide transport and alternative oxidase activity thus suggesting a perturbation of cell wall
synthesis and respiration. Furthermore, KP susceptibility of a range of C. albicans mutants,
including deltacnh1, deltatok1, deltachs2, deltachs8, deltatps1, deltacap1 and deltadit2 was also
investigated. Investigations have revealed that all of these C. albicans mutants exhibit altered
sensitivity to KP thus suggesting that the deficient proteins are required for (i) the activity of KP
or (ii) the cellular response to KP. Prior exposure of cells to ion channel inhibitors was also found
to alter the sensitivity of Candida cells to KP thus suggesting that KP may induce osmotic stress.
While KP is believed to associate with the cell wall and cell membrane of C. albicans, using KP
conjugated with flourescein we have observed that KP is internalized thus providing evidence that
KP might act on an intracellular target.




                                                 131
P82A
The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in
complex with pepstatin A
Olga Hruskova-Heidingsfeldova1, Jiri Dostal1, Jiri Brynda2, Irena Sieglova2, Iva Pichova1 and
Pavlina Rezacova2
1 Gilead Sciences Research Centre, Institute of Organic Chemistry and Biochemistry, Flemingovo
nam. 2, Prague 6 166 10, Czech Republic, Phone: +420 220 183 249, FAX: +420 224 310 090, e-
mail: olga-hh@uochb.cas.cz, Web: www.uochb.cas.cz
2 Institute of Molecular Genetics, Flemingovo nam.2, Prague 6, Czech Republic

Secreted aspartic proteinases of pathogenic Candida spp. are considered as one of the virulence
factors. Candida parapsilosis possesses three genes encoding these enzymes: SAPP1-3. We have
purified the Sapp1p isoenzyme from the C. parapsilosis culture medium, and determined its three-
dimensional crystal structure in complex with pepstatin A, the classical inhibitor of aspartic
proteinases. Overall fold and topology of Sapp1p is similar to the archetypical fold of monomeric
aspartic proteinase family and known structures of Sap isoenzymes from C. albicans and Sapt1p
from C. tropicalis. Structural comparison revealed noticeable differences in the structure loops
surrounding the active site. This resulted in differential character, shape, and size of the substrate-
binding site explaining differential substrate specificities and inhibitor affinities.
In comparison with the Sapp1p sequence deposited in NCBI, our structure harbors three naturally
occurring mutations: Leu193 to Ser, Pro196 to Ala and deletion of Tyr between residues 312 and
313. The exchange of Leu193 to Ser is due to the CUG codon usage. Naturally occurring Pro to
Ala exchange might not be unique to Sapp1p. It was found also in beta-1,3-glucan synthase Fks1p
of C. parapsilosis and was associated with a reduced susceptibility of C. parapsilosis towards the
echinocandin-class antifungals in comparison with C. albicans (Garcia-Effron et al., (2008)
Antimicrob Agents Chemother 52, 2305). In the Sapp1p structure, strikingly, both Leu193 - Ser
and Pro196 - Ala are positioned on a short loop (192-197 loop), proximal to P3’ residue of
pepstatin A bound in the active site. Although P3’ residue of the inhibitor does not play a crucial
role in the interaction with the enzyme, the effect of naturally occurring mutations within the 192-
197 loop has to be elucidated. To date, we have found that the Leu193 to Ser exchange does not
significantly affect the enzyme kinetics of Sapp1p. The role of Pro196 - Ala is under investigation.
This work was supported by the Czech Science Foundation (grant 310/09/1945) and by the
Ministry of Education of the Czech republic (grant LC 531).




                                                 132
P83B
Gain of function mutations in CgPDR1 of C. glabrata not only mediate antifungal resistance
but also enhance virulence
Sélène Ferrari1, Françoise Ischer1, David Calabrese1, Brunella Posteraro2, Maurizio Sanguinetti2,
Giovanni Fadda2, Bettina Rohde3, Christopher Bauser3, Oliver Bader4 and Dominique Sanglard1
1 Institute of Microbiology, University of Lausanne and University Hospital Center, Bugnon 48,
Lausanne 1011, Switzerland, Phone: +41213144062, FAX: +41213144060, e-mail:
selene.ferrari@chuv.ch
2 Institute of Microbiology, Università Cattolica Sacro Cuore, Roma, Italy.
3 GATC Biotech AG, Konstanz, Germany.
4 Institut für Medizinische Mikrobiologie, Universitätskliniken Göttingen, Göttingen, Germany.

C. glabrata develops azole resistance mostly via the upregulation of ABC transporter genes
CgCDR1, CgCDR2 and CgSNQ2. CgPdr1p is the major C. glabrata transcription factor involved
in their regulation. Gain of function (GOF) mutations in CgPDR1 are responsible for the increased
expression of CgCDR1, CgCDR2 and CgSNQ2 and thus to contribute to azole resistance of
clinical isolates.
In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical
isolates and tested their relevance not only to azole resistance in vitro and in vivo but also to
virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched
isolates (n=122) enabled the identification of 57 amino acid substitutions present only in CgPDR1
alleles from azole-resistant isolates. These mutations are GOF mutations since only alleles
containing these mutations conferred ABC-transporter genes constitutive high expression.
Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2 and
CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF
mutations, suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting
elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-
transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 GOF alleles were
not only more virulent in mice than those with wild type alleles, but they also gained fitness in the
same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole
treatment failure in the mouse model.
This study shows the high variability in CgPDR1 GOF mutations having differentiated effects on
target genes including the major ABC-transporters involved in azole resistance. Importantly, this
study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo
azole resistance but that they can also confer a selective advantage under host conditions.




                                                133
P84C
Functional dissection of Tac1p, a Candida albicans transcription factor invoved in antifungal
drug resistance
Alix T. Coste, Jérôme Crittin, Vincent Turner and Dominique Sanglard
Institute of Microbiology of the University of Lausanne, University of Lausanne,University
Hospital Center, Bugnon, 48, Lausanne 1011, Switzerland, Phone: +41 (0)21 314 40 61, FAX:
+41 (0)21 314 40 60, e-mail: alix.coste@chuv.ch, Web: http://www.chuv.ch/imul

For treating C. albicans infections, repetitive use of antifungal agent including azoles leads to an
adaptation of the fungus to the drug pressure and eventually to drug resistance. Resistance
mechanisms fall into different categories but one of the most frequent is the increased drug efflux
through enhanced expression of the ABC (ATP-Binding Cassette)-transporters CDR1 (Candida
Drug Resistance) and CDR2. The understanding of the transcriptional regulation of these genes is
therefore critical for the control of azole resistance through efflux mechanisms. The transcriptional
regulation of CDR1 and CDR1 is mediated by TAC1, a Zn2Cys6transcription factor, which is able
to bind to the promoters of its target genes. We previously identified TAC1 hyperactive alleles
(TAC1-hyp) from clinical azole-resistant strains, which in contrast to wild-type alleles (TAC1-
wt), conferred constitutive high CDR1/CDR2 expression in a tac1 deletion mutant. Hyperactivity
of TAC1 are due to gain-of-function mutations (GOF). A better knowledge of Tac1p and its
partners will lead to a better understanding of the azole resistance phenomenon. In this work we
functionally dissected this protein. For this purpose, tagged versions of Tac1p were constructed to
performed immuno-precipitation of distinct Tac1p forms (wt or hyp). Our results demonstrated
that Tac1p could form dimers. Chromatin immuno-precipitation (ChIP) assays allow to
established that Tac1p binds intrinsically to target promoters indicating the role of potential
partners to activate the transcription of Tac1p target genes. Finally, we established functional
regions of the protein by deleting the putative DNA binding domain, the putative transcriptional
inhibitory and activation domains. Functionality of the mutant proteins were analysed with 2
different systems. Our results showed that the last 60 aa of Tac1p were sufficient to allow
transcriptional activity. Nevertheless, optimal activity was obtained using the last 160 aa. In
contrast, a region including the last 240 of the protein led to complete loss of transcriptional
activity and thus suggests the presence of a transcriptional inhibitory domain located upstream of
the last 160 aa of the protein. Further constructions are currently designed and tested to determine
the role of other Tac1p regions in the activation of target genes transcription.




                                                134
P85A
Genome-wide gene expression profiles of individual CgPDR1 hyperactive alleles and
identification of CgPdr1p-dependent virulence factor(s) in Candida glabrata
Sélène Ferrari and Dominique Sanglard
Institute of Microbiology, University of Lausanne and University Hospital Center, Bugnon 48,
Lausanne 1011, Switzerland, Phone: +41213144062, FAX: +41213144060, e-mail:
selene.ferrari@chuv.ch

CgPdr1p is a C. glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of ABC-
transporter genes (CgCDR1, CgCDR2 and CgSNQ2) mediating azole resistance. By comparison
of CgPDR1 alleles from azole-susceptible and azole-resistant related clinical isolates, we observed
a high diversity among CgPDR1 alleles and identified 57 distinct single amino acid (aa)
substitutions conferring hyperactivity to CgPdr1p and high expression of ABC transporter genes.
Although CgCDR1, CgCDR2 and CgSNQ2 are all regulated by CgPdr1p, they are not always co-
ordinately expressed in azole-resistant isolates indicating that ABC transporter genes were
differentially regulated depending on the mutation present on individual CgPDR1 alleles.
Moreover, the aa substitutions in CgPdr1p enhance virulence and lead to fluconazole treatment
failure in mouse models. Taken together these data demonstrate a high variability in CgPDR1
mutations, which themselves have differentiated effects on target genes including ABC-
transporters and probably on yet unidentified virulence factors.
In this study, we aimed to determine genome-wide changes in gene expression driven by seven
individual CgPDR1 hyperactive alleles as compared to wild-type allele to identify i) the CgPdr1p
target genes differentially expressed in presence of CgPDR1 hyperactive alleles and ii) potential
virulence factor(s) regulated by CgPDR1 hyperactive alleles. Microarray experiments revealed a
high number of genes (ranging from 80 to 400 genes) differentially regulated by individual
CgPDR1 hyperactive alleles. Enrichment of specific biological processes (stress response,
resistance to DNA damage and cell wall biogenesis) was observed upon expression of specific
CgPDR1 alleles. These processes may contribute individually or in combination to modulate
virulence of C. glabrata. Consistent with previous observations, we observed a poor overlap in the
number of co-ordinately expressed genes from all hyperactive alleles. Only two genes were
commonly upregulated by all tested hyperactive alleles. Since the CgPDR1 hyperactive alleles
used in this study were shown to enhance C. glabrata virulence in animal models, our current
studies are addressing the involvement of these two genes in azole resistance and virulence.




                                               135
P86B
Molecular analysis of the Candida albicans multidrug resistance regulator MRR1
Sabrina Schubert1, P. David Rogers2 and Joachim Morschhäuser1
1 Institut für molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, Würzburg
97070, GERMANY, Phone: +49 931 31 2127, FAX: +49 931 31 2578, e-mail: s.schubert@uni-
wuerzburg.de, Web: http://www.infektionsforschung.uni-wuerzburg.de/
2 University of Tennessee Health Science Center, Memphis, Tennessee, USA

Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major
facilitator superfamily, is a frequent cause of resistance to the widely used antimycotic agent
fluconazole and other toxic compounds in Candida albicans. The zinc cluster transcription factor
Mrr1 controls MDR1 expression in response to inducing chemicals, and gain-of-function
mutations in MRR1 are responsible for the constitutive MDR1 upregulation in all clinical and in
vitro generated fluconazole-resistant C. albicans strains tested so far. In order to understand how
Mrr1 activity is regulated, we aimed at identifying the functional domains of this transcription
factor. MRR1 alleles with serial C-terminal deletions were tested for their ability to induce the
MDR1 promoter in a C. albicans reporter strain lacking the endogenous MRR1 alleles, thereby
delimiting the minimal size of Mrr1 that is essential for its activity. In addition, internal Mrr1
fragments were fused to the tetracycline repressor and tested for their ability to induce
transcription from a tetR-dependent promoter. By fusing Mrr1 fragments to the Gal4 transcription
activation domain we investigated if the N-terminally located DNA-binding domain of Mrr1 was
sufficient to specifically activate the MDR1 promoter and confer drug resistance. The results of
these analyses contribute to a detailed understanding of the function of an important regulator of
drug resistance in C. albicans.




                                               136
P87C
Heterologous expression of the Candida albicans plasma membrane proton pump in
Saccharomyces cerevisiae
Mikhail Keniya1, Ann Holmes1, Masakazu Niimi2, Richard Cannon1 and Brian Monk1
1 Oral Sciences, University of Otago, 310 Great King Street, Dunedin 9016, New Zealand, Phone:
+64 (3) 479 3873, FAX: +64 (3) 479 7078, e-mail: mikhail.keniya@otago.ac.nz
2 Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo, Japan

Candida albicans remains the dominant cause of both opportunistic fungal infections and life-
threatening systemic infections, especially in the immunocompromised. The plasma membrane
proton pumping ATPase (Pma1p), an essential enzyme that generates the electrochemical gradient
required for nutrient uptake and ionic homeostasis, is a validated target for new antifungals. The
expression of CaPma1p in the model yeast Saccharomyces cerevisiae should facilitate screening
for Pma1p inhibitors and structure-directed antifungal discovery.
The PMA1 ORF in a haploid S. cerevisiae strain that is hypersensitive to xenobiotics was replaced
with the homologous ORF from C. albicans, together with an N-terminal hexa-His tag, the strong
PGK terminator and a downstream URA3 marker. The growth of transformants expressing
CaPma1p was inhibited at low pH and chimeric suppressor mutants arose as a result of
recombination between CaPMA1 and the homologous, but non-essential, ScPMA2 gene. Deletion
of ScPMA2 circumvented this problem. The identity of the CaPma1p band (~100 kDa)
heterologously expressed in the S. cerevisiae plasma membrane was confirmed by mass
spectrometry. The enzyme was found to be expressed at significantly lower levels and have lower
specific activity than ScPma1p in the parent strain. Heterologously expressed CaPma1p had
properties (pH and temperature optima, and response to inhibitors) that more closely resembled
ScPma1p than native CaPma1p, possibly due to environmental factors or post-translational
modification in S. cerevisiae. Analysis of suppressor mutants revealed that specific residues
between aa 531-595 of CaPma1p may affect the formation of intermolecular complexes of Pma1p
in S.cerevisiae. Failure to make hexameric structures by CaPma1p may result in its mislocalization
or enhanced degradation.
The amount of active CaPma1p expressed in the S. cerevisiae host correlated with sensitivity to
the translation inhibitor hygromycin B. The uptake of this drug is dependent on the membrane
potential generated by Pma1p. Preliminary experiments indicate that the strains differentially
expressing CaPma1p can be used in screens of compound libraries to identify high affinity
inhibitors of CaPma1p.
This research was supported by NIH grants DE015075 and DE016885, and the NZ Lottery Grants
Board.




                                               137
P88A
URA3 status of a null mutant of RTA2, a calcineurin pathway gene, affects its phenotype
Sarmistha Mahanty and Sneh Lata Panwar
School of Life Sciences, Jawaharlal Nehru University, New Mehrauli Road, New Delhi 110067,
INDIA, Phone: +91 (11) 2670 4620, FAX: +91 (11) 2674 2588, e-mail:
sneh@mail.jnu.ac.in,sneh10@hotmail.com

RTA2 (Resistance to 7-aminocholesterol) is described as a putative phospholipid translocase in
CGD bearing homology to the RSB1 and RTA1 genes from S. cerevisiae. Microarray experiments
suggest this gene to be one of the target genes of the calcineurin-responsive transcription factor,
CRZ1 in C. albicans. In lieu of the background of the role that the orthologues of RTA2 play in S.
cerevisiae and the role of calcineurin pathway in membrane stress, in this study we have explored
the function of RTA2 in C. albicans. For the analysis, we first constructed a null mutant of RTA2
in the strain RM1000 using the HIS1 and the "URA3-blaster" cassettes. We observed differences
in phenotypes between the rta2 null mutant, which was URA3+ (SM1; rta2delta::hisG-URA3-
hisG/rta2delta::HIS1) and the rta2 null mutant which was URA3- (SM2;
rta2delta::hisG/rta2delta::HIS1). While SM1 and SM2 both were sensitive to 7-aminocholesterol
(7-AC), which is known to be a specific substrate for RTA1 in S. cerevisiae, their ability to grow
in the presence of azoles differed. We noticed that SM1 displays an increased resistance to
fluconazole, ketoconazole and terbinafine while SM2 does not show any alteration in growth on
azoles. Furthermore, there is no phenotype for SM1 in presence of the long chain base,
phytosphingosine (PHS), while the heterozygous strain displays sensitivity to PHS. Membrane
permeability assays using various methods indicate changes in the membrane of both SM1 and
SM2, pointing to a role for RTA2 in maintaining a proper plasma membrane environment in C.
albicans, which when altered could be affecting azole susceptibilities. To begin with, the
membrane permeability of the two wild type strains, CAF2-1 and RM1000, differ from each other.
Taken together, our results show that maybe the URA3 selectable marker in SM1 by some
unexplained means so far, specifically affects the azole phenotype of this strain. Our assumption
from the results so far is that the ectopic expression of URA3 might be responsible for this
difference in phenotype. Our preliminary results are indicative of effects that the positioning of
URA3 gene cause on drug profilings. The role of URA3 marker in affecting the drug phenotype is
currently being assessed in our laboratory.




                                               138
P89B
Properties of the autoactive-truncated form of Cmp1p, the catalytic subunit of calcineurin of
Candida albicans
Vincent Turner and Dominique Sanglard
Insitute of Microbiology, University of Lausanne & University Hospital Center, Bugnon 48,
Lausanne 1011, Switzerland, Phone: +41 21 314 40 62, FAX: +41 21 314 40 60, e-mail:
vincent.turner@chuv.ch

Azole antifungals possess a fungistatic activity in Candida albicans and make this yeast tolerant to
these agents. The fungistatic properties of azoles may have facilitated the ability of C. albicans to
develop drug resistance. Thus, the conversion of azoles into fungicidal agents is of interest. In C.
albicans, the Ca2+-activated phosphatase calcineurin (CN), which is composed of a catalytic
(CMP1) and a regulatory (CNB1) subunit, is essential for azole tolerance. CN activity is under the
control of several regulatory features i) CN is a dimeric protein; ii) CN is expressed as a non-
active form and iii) CN is activated by Ca2+ (which has CN-independent side-effects on the
transcriptome). To bypass these regulatory features, an autoactive-truncated form of CMP1
(CMP1tr) was engineered to mimic the properties of the wild type active form of CN.
This work is aimed to characterize in C. albicans i) the ability of Cmp1trp to mimic a wild type
active form of CN and ii) the dependence of Cmp1trp to Ca2+. For this purpose, C. albicans
strains were designed for expressing CMP1 and CMP1tr alleles in a doxycycline-dependent
manner.
In that study, we revealed that Cmp1trp behaved as a wild type CN for the following processes: i)
Cmp1trp was able to dephosphorylate the CN-dependent transcription factor CaCrz1p ii) the
dephosphorylation of CaCrz1p by Cmp1trp led to the CaCrz1p-dependent expression of RTA2 iii)
the activity of Cmp1trp was inhibited by the CN inhibitor cyclosporine A.
In a second time, we showed that the expression of Cmp1trp had no influence on C. albicans
growth in rich medium. However, only the strain expressing Cmp1trp was able to grow on a
medium supplemented with 4mM BAPTA (Ca2+ chelator) otherwise inhibiting the growth of the
wild type. This indicates that expression of Cmp1trp compensates for low Ca2+ concentration in
the growth medium. The exposure of strains to Ca2+ did not hyperactivate Cmp1trp as shown
with expression analysis against RTA2 otherwise activating the wild type. This means that
Cmp1trp as probably an activity independent of external Ca2+ concentrations.
In conclusion, this study showed that the activity Cmp1trp was independent from Ca2+. In
addition, it demonstrated that Cmp1trp could behave as a wild type active form of CN. According
to our results, Cmp1trp is an interesting tool to investigate the role of CN in C. albicans.




                                                139
P90C
Adenosine decreases phagocytosis of Candida albicans by RAW 264.7 cells
Carolina Coelho1, Filipa Curado1, Vitor Cabral1, Rodrigo Cunha2 and Teresa Gonçalves1
1 Medical Mycology Yeast Research Group, Center for Neuroscience and Cell Biology,
Faculdade de Medicina Universidade de Coimbra, Coimbra 3004-504, Portugal, Phone: +351 239
857772, FAX: +351 239 822776, e-mail: filipa.curado@gmail.com
2 Purines at CNC, CNBC, Coimbra, Portugal

Macrophages have a primordial role in the host immune response to Candida albicans infection,
but this yeast has developed strategies to overcome this initial line of defence by mechanisms still
unsolved. This knowledge is of major importance to the development of novel and effective anti-
fungicide strategies. This work was devised to test the novel hypothesis that purines, particularly
adenosine, and their sensing devices may constitute a key system exploited by C. albicans to
evade macrophage attack, thus explaining its success as a pathogen. The extracellular catabolism
ATP by ecto-nucleotidases, which are ubiquitous, yields adenosine, which is the major stop signal
of the immune system in general and of macrophages in particular (Ohta & Sitkovsky. 2001.
Nature, 414: 916-920).
Our first approach to this aim was to test whether adenosine and 2-chloro-adenosine, a non-
metabolizable analogue of adenosine, influenced the phagocytic efficiency of macrophages. The
phagocytic rate was studied in a macrophage derived cell line, RAW 264.7 cells, using a
differential fluorescence microscopy methodology with Oregon Green (labelling all yeast cells),
and Calcofluor White (only labelling non-ingested yeast cells) (Fernandez-Arenas et al. 2007. Mol
Cel Proteomics 6:460-478).
Adenosine and 2-chloro-adenosine had no direct effect on either the viability or morphology of
yeast cells; in particular, it did not affect C. albicans cells. Nevertheless, the infection of RAW
264.7 cells with C. albicans (ratio 1:1) resulted in a decreased phagocytic rate in the presence of
adenosine 10 microM, whereas 2-chloro-adenosine was devoid of effect. The results obtained
indicate that adenosine exerts its function as a STOP signal for the immuno-inflammatory system
also in the case of fungal infections.




                                                140
P91A
Reverse genetics in the human fungal pathogen C. albicans aiming at improving current
drug treatment options
Elias Epp, Doreen Harcus, Anna Lee, Jamie Surprenant, Gregor Jansen, Michael Hallet, David
Thomas and Malcolm Whiteway
Biology, McGill University, 1205 Docteur Penfield, Montréal H3A 1B1, CANADA, Phone: 001
514 496 1529, FAX: 001 514 496 6213, e-mail: elias.epp@mail.mcgill.ca

Candida albicans is among the leading causes of mycoses-related deaths and both a limited
availability of antifungal drugs as well as an emergence of drug resistance feed into a general
public health concern. One of the most widely used drugs to treat C. albicans infection is the
fungistatic drug Fluconazole (FCZ). Here we aimed at improving the nature of FCZ by making it
fungicidal through combination with other drugs. To this end, we have first relied on
Saccharomyces cerevisiae. By screening the yeast knock out collection of 4700 non-essential
genes for mutants that cannot survive in the presence of FCZ, we have found a robust set of 23
genes (FCZ-fungicidal genes), that can be sorted into 5 groups: SAGA complex mutants, V-
ATPase mutants, cell wall/cytoskeletal mutants, mediator complex mutants and others. Next, we
attempted to confirm the yeast prediction by knocking out the homologous genes in C. albicans
and testing these mutants for FCZ-fungicidality. Surprisingly, we only found one case where the
behaviour in C. albicans corresponded to the S. cerevisiae prediction. Only Gcs1p, an ARF GAP
(ADP-ribosylation factor GTPase activating protein), becomes essential for survival in presence of
FCZ in both S. cerevisiae and C. albicans. Knocking out GCS1 in C. albicans FCZ-resistant
clinical isolates restored FCZ sensitivity. We then confirmed chemically the GCS1-depenent FCZ
sensitivity by showing a potent synergy between Brefeldin A (BFA), an ARF GEF [guanine
nucleotide exchange factor (GEF) for ADP ribosylation factors (ARF)] inhibitor, and FCZ in WT
C. albicans as well as in FCZ-resistant clinical isolates.
Finally, we attempted to confirm the FCZ/BFA synergy found in vitro in a mouse model of
disseminated candidiasis. In vivo studies showed that the C. albicans gcs1 mutant is avirulent in
B6 and attenuated in virulence in an A/J mouse model. The anticipated FCZ/BFA in vivo synergy
is currently being tested in the B6 model.




                                               141
P92B
Role of the C. albicans ortholog of yeast GCS1 in multidrug resistance and hyphal growth
Thomas Lettner, Ute Zeidler, Michael Breitenbach and Arnold Bito
Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, Salzburg 5020,
Austria, Phone: +43 (0)662 8044 5793, FAX: +43 (0)662 8044 144, e-mail: arnold.bito@sbg.ac.at

In a systematic study of the phenotypic consequences of several C. albicans deletion mutants, the
null mutant of the homolog (orf19.3683) of the S. cerevisiae gene GCS1 showed a very high
sensitivity to several unrelated toxic compounds. These included therapeutic drugs like
miconazole, itraconazole and hygromycin B. Although the gene name "GCS1" has been given to
the ortholog of the S. cerevisiae GSH1 gene in the Candida Genome Database, we will use it for
the ortholog of ScGCS1 here. The ScGcs1 protein has been shown to function as an Arf GTPase-
activating protein (Arf-GAP) and is required for several pathways of intracellular vesicle and
protein traffic, e.g. from the ER to the Golgi apparatus and to the cytoplasmic membrane but also
for endocytic vesicles to the vacuole. Several drug transporters have been identified in C. albicans
and found to be required for the high tolerance of this fungal pathogen to several classes of toxic
compounds. These transporters are membrane-spanning proteins located in the cytoplasmic or
vacuolar membranes. Therefore, assuming that the C. albicans protein has the same biochemical
function as its yeast homolog, the compound susceptibility of Candida gcs1 null mutant cells may
be caused by inefficient transport of vesicles carrying drug transporters to the cytoplasmic and
vacuolar membranes and subsequent lower amount of these proteins at their functional sites.
Several results of the ongoing study are consistent with a function of CaGcs1p as an Arf-GAP and
in vesicle traffic. A Gcs1-GFP fusion protein was found to be localized throughout the cytoplasm.
The mutant strain showed a delay in endocytic uptake of the fluorescent dye FM4-64. Compared
to the wild-type, the mutant strain had a 10-fold higher susceptibility to brefeldin-A which inhibits
the GTPase activity of Arf proteins. Moreover, upon hyphal induction gcs1 mutant cells
inefficiently formed true hyphae which showed morphological defects. Experiments are under
way with the aim to confirm i) direct interaction of CaGcs1p and CaArf1p at the protein level, ii)
inappropriate intracellular localization of a few GFP-tagged drug transporters in gcs1 mutant cells,
and iii) that the C. albicans GCS1 gene is able to complement the phenotypic defects of the S.
cerevisiae gcs1 null mutant. The results will be presented.




                                                142
P93C
Adrenaline Promotes Fluconazole Efflux on Candida albicans
Sofia Costa de Oliveira1, Ana Silva Dias1, Cidalia Pina-Vaz1, Daniel Moura2 and Acacio G
Rodrigues3
1 Microbiology, Porto Faculty of Medicine, Alameda Prof. Hernani Monteiro, Porto 4200-319,
Portugal, Phone: +351 225513662, FAX: +351 225513662, e-mail: sqco@med.up.pt, Web:
http://www.med.up.pt
2 Institute of Pharmacology and Therapeutics
3 Burn Unit, Department of Plastic and Reconstructive Surgery, Faculty of Medicine, University
of Porto

Vasoactive amines are frequently prescribed as life saving medication to critical care patients.
Clinical antifungal resistance might result from concomitant administered drugs, which may
change the susceptibility pattern by triggering resistance mechanisms. FUN-1 and Rhodamine 6G
(Rh-6G) are fluorescent substracts of efflux pumps which have been shown to be over expressed
in azole resistant Candida strains (1,2).
The aim was to evaluate the effect of adrenaline upon the extrusion of fluconazole by Candida
albicans efflux pumps.
Six fluconazole susceptible C. albicans strains, were studied. Susceptibility testing to fluconazole
was performed accordingly CLSI reference protocol and re-determined in the presence of
adrenaline. Drug interactions were studied using the checkerboard procedure.
To quantify the efflux by flow cytometry, blastoconidia suspensions were incubated with growing
concentrations of adrenaline for 90 minutes and afterwards stained with 0.5 µM FUN-1 and with
Rh-6G. The mean intensity of fluorescence was registered and the percentage of reduction of
staining calculated. In control assays, 3 C. albicans strains with selective deletion of efflux pumps
genes DSY 448 (cdr1/cdr1 deleted) (3), DSY 653 (cdr2/cdr2 deleted) (4) and DSY 654 (cdr1/cdr2
deleted) (4) (kindly gifted by Prof. D. Sanglard) were tested accordingly the above described
experimental protocol.
The median FIX for fluconazole and adrenaline was 5 (2-17) (antagonistic effect). Candida
blastoconidia exposed to adrenaline showed a dose dependent decrease in the intensity of FUN-1
or Rh6G staining compared with non-treated cells. Following incubation with adrenaline, the
percentage of reduction of FUN-1 and of Rh-6G staining was higher in DSY 653 than in DSY
448, although lower than the reduction observed with clinical strains. No decrease of staining was
observed in DSY 654 strain.
Adrenaline increases the activity of efflux pumps in C. albicans strains, leading to the extrusion of
FUN-1 and Rh-6G. Such results strongly support the concept that concomitant therapy with
inotropic amines might reduce azole susceptibility of fungal isolates from patients receiving
adrenaline infusions.

1- Pina-Vaz, C, et al., 2000, J Med Microbiol, 49: 831-840.
2- Coste, A, et al., 2006, Genetics, 4: 2139-2156.
3- Sanglard, D, et al., 1996, Antimicrob Agents Chemother, 40: 2300-2305.
4- Sanglard D, et al., 1997, Microbiology, 143: 405-416.




                                                143
P94A
Candida albicans and Candida parapsilosis: divergence on azole resistance mechanisms
Elisabete Ricardo, Ana Pinto Silva, Sofia Costa-de-Oliveira, Acácio Gonçalves Rodrigues and
Cidália Pina-Vaz
Microbiology, Faculty of Medicine, Alameda Prof Hernani Monteiro, Porto 4200-319, Portugal,
Phone: +351 225513662, FAX: +351 225513662, e-mail: betaricardo@yahoo.com

Fungal infections represent a common but serious problem in public health, being responsible for
high morbidity and mortality. In a recent epidemiological survey performed by our team at a
Portuguese University Hospital, C. albicans and C. parapsilosis were the most frequent fungal
isolates (1). There is a growing concern about antifungal drug resistance, being the overexpression
of efflux pumps encoded by CDR1, CDR2 and MDR1 genes one of the most well characterized
azole resistance molecular mechanisms displayed by C. albicans, but short knowledge is yet
available regarding C. parapsilosis.
In order to evaluate the role of ATP-dependent efflux pumps on antifungal resistance in C.
albicans and in C. parapsilosis, and corroborate ibuprofen reverting activity on C. albicans
resistant strains (2), phenotypic assays were performed involving three resistant strains of each
species. We determined the MIC values to the azole drugs fluconazole, voriconazole and
posaconazole, accordingly the CLSI M27-A3 protocol in the absence and presence of ibuprofen
100 mg/L (described as efflux blocker) (2). Also, agar disk diffusion assay was performed with
another efflux blocker FK506 (Tacrolimus) (ten-fold dilutions ranging from 100 to 0.1µg/ml) as
described by Onyewu et al. (2003), with some alterations: FK506 is prepared in DMSO;
fluconazole was added at supra-MIC value (128 µg/ml) to YEPD agar plates, in which FK506
disks were placed. Additionally, flow cytometric assays using rhodamine 6G (Rd-6G) 5 µM, an
efflux pumps fluorescent substrate, were performed in order to confirm the efflux activity,
according to Sanglard et al, (1999) without and with ibuprofen.
In the presence of ibuprofen and FK506 a synergistic effect was observed for C. albicans resistant
strains. Regarding C. parapsilosis no synergistic effect between ibuprofen or FK506 and azoles
was detected. The cytometric studies showed that only C. albicans displayed an increase in Rd-6G
staining in the presence of ibuprofen.
The present data shows the importance of ATP-dependent efflux on C. albicans resistant strains,
in contrast to C. parapsilosis where efflux seems to play a minor role in azole resistance.
Ibuprofen seems quite promising in the treatment of infections by azoles, on resistant C. albicans
strains. Regarding C. parapsilosis further studies need to be undertaken.

1. Costa-de-Oliveira S. et al. (2008) Eur J Clin Microbiol Infect Dis. 27:365
2. Pina-Vaz C. et al. (2005) J Antimicrob Chemother. 56:678




                                                144
P95B
Morphogenic regulator Efg1p of Candida albicans affects drug susceptibilities independent
of drug efflux pumps
Tulika Prasad1, Saif Hameed2, Chinmay K. Mukhopadhyay3, Sudipta Biswas3, Eleonora R.
Setiadi4, Joachim F. Ernst4 and Rajendra Prasad2
1 Advanced Instrumentation Facility, University Science Instrumentation Centre, Jawaharlal
Nehru University, New Delhi 110067, India, Phone: +91-11-26704560, FAX: +91-11-26741081,
e-mail: prasadtulika@hotmail.com, Web: www.geocities.com/ResearchTriangle/lab/5540/
2 Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New
Delhi-110067, India
3 Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi-110067, India
4 Institut für Mikrobiologie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany

In this study, we show that null mutants of the morphogenic regulator EFG1 are sensitive to drugs
particularly to those targeting ergosterol or its metabolism. Efg1p disruption showed a gene
dosage effect on drug susceptibilities and resulted in greater sensitivity to azoles and polyenes in
homozygous mutant as compared to the wild type, heterozygous and revertant with one allele
retransformed strains. Northern experiments and microarray data with delta efg1 null mutants
ruled out involvement of genes encoding major drug efflux pumps such as CDR1, CDR2 and
CaMDR1. This was further confirmed since we did not find any change in the R6G and
methotrexate efflux (specific substrates of CDR1 and CaMDR1 respectively) in delta efg1 null
mutants. However, we observed that delta efg1 null mutants displayed increase in membrane
fluidity which coincided with down regulation of ERG11 and a simultaneous up regulation of
OLE1 and ERG3. These changes in gene expression resulted in a 2 fold increase in oleic acid and
23 % lowering of ergosterol contents in delta efg1 null mutants. Our results demonstrate that these
lipid compositional changes led to an increase in passive diffusion of drugs. Additionally,
increased levels of ROS (Reactive Oxygen Species) in delta efg1 null mutants was experimentally
showed by biochemical and fluorescent methods of analysis and microarray data supported by
Northerns were found to be coupled with down regulation of oxidative stress response genes
namely, RBT5, SOD2, CTA1, DDR48 and GRP2. To summarize, our data revealed that increased
levels of ROS and related enzymes as well as increased membrane fluidity due to altered
membrane lipid composition could be important determinants in contributing to the increased drug
susceptibility of the delta efg1 null mutant cells.




                                                145
P96C
Frequency of azole resistance phenotypes in the two most prevalent human pathogenic
yeasts C. albicans and C. glabrata
Oliver Bader1, Martin Kuhns1, Claire Martel2, Josie Parker2, Kathrin Tintelnot3, Michael Seibold3,
Emilia Mellado4, Marie-Elisabeth Bougnoux5, Christophe d’Enfert5, Dominique Sanglard6, Diane
Kelly2, Steve Kelly2, Uwe Gross1 and Michael Weig1
1 Institute for Medical Microbiology, University Göttingen, Kreuzbergring 57, Göttingen 37075,
Germany, Phone: +49 (551) 39 22346, FAX: +49(551) 39 5861, e-mail: obader@gwdg.de
2 Institute of Life Science and School of Medicine, Swansea University, Swansea, Wales, UK
SA2 8PP
3 Mycology Department, Robert Koch-Institute, 13353 Berlin, Germany
4 Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, 28.220
Majadahonda, Madrid, Espana
5 Unité Postulante Biologie et Pathogénicité Fongiques, Institut Pasteur, 75724 Paris Cedex 15,
France
6 Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland

We have investigated the resistance phenotypes of a collection of over 200 clinical C. albicans
and C. glabrata isolates with increased tolerance towards azole antifungal drugs. Although the
range of MIC values for Voriconazole is generally 100-fold lower than the one for Fluconazole,
MIC testing (EUCAST) of the strains revealed that there is a clear linear relation of Fluconazole
and Voriconazole tolerance. Interestingly, we did not observe any Fluconazole resistant strains
that did not have elevated Voriconazole tolerance. In contrast, there were several C. albicans
strains which showed Voriconazole resistance, but were still susceptible to Fluconazole. To
elucidate the mechanisms underlying these patterns, all strains were tested for unspecific drug
efflux (Rhodamine6G accumulation) and for the membrane sterol composition (gas
chromatography). For C. glabrata the analysis shows that resistance is mediated almost
exclusively by drug efflux, only two isolates were found which showed the phenotype of an
ERG11 mutation. In contrast, C. albicans showed a large variety of different sterol compositions,
indicating different mutations in the ergosterol biosynthesis pathway. The majority of cross-
resistant strains showed drug efflux, many in combination with a sterol composition phenotype of
an ERG11 mutation. Strains which were exclusively Voriconazole resistant mediated this only by
changes in sterol composition. Also, a series of mutants exhibiting altered sterol C5-desaturation
(encoded by ERG3) with infinite azole resistance were identified. Additionally, these isolates
showed a linear correlation of ergosterol content and Amphotericin B resistance. The molecular
reasons for the phenotypes described here as well as the phylogenetic relationships of the strains
are currently under investigation. In conclusion, we were able to identify phenotypical changes
leading to increased azole drug tolerance for the majority of resistant isolates in our collection.
However still a few strains remained for which no potential resistance mechanism could be
postulated.




                                               146
P97A
Antifungal activity and mode of action of ApoEdpL-W, a cationic peptide derived from the
human apolipoprotein E
Tristan Rossignol1, Curtis Dobson2 and Christophe D'Enfert1
1 Unité Biologie et Pathogénicité Fongiques, Institut Pasteur, 25, rue du Docteur Roux, Paris
75015, France, Phone: +33(0)145688205, FAX: +33 (0)1 45 68 89 38, e-mail:
tristan.rossignol@pasteur.fr, Web: http://www.pasteur.fr/bpf
2 Ai2 Ltd, G Floor, The Mill, Sackville Street, Machester M60 1QD, United Kingdom

A novel class of peptides derived from human apolipoprotein E (apoE) with a broad-spectrum
anti-microbial activity has recently been reported. In this study we have investigated the antifungal
properties of the ApoEdpL-W peptide (WRKWRKRWWWRKWRKRWW), a simple highly
cationic peptide derivative of apoE and subsequently attempted to understand its mode-of-action.
MIC of ApoEdpL-W towards C. albicans strain SC5314 was determined by broth dilution method
and was in the range of 5 µM. While incubating C. albicans at ApoEdpL-W concentrations equal
or below MIC was fungistatic, incubation at concentrations above MIC was fungicidal, no viable
cells being detected after an hour of exposure.
The activity of ApoEdpL-W was also tested at different steps of biofilm formation using a 96 well
plate model. Addition of the peptide at a final concentration of 5 µM immediately after adhesion
(i.e. prior to biofilm formation) resulted in a growth inhibition similar to that observed with a
planktonic culture. In contrast, when the peptide was added at a later time point, i.e. when the
biofilm had already developed, biofilm formation was reduced by 50 %. Increasing peptide
concentrations above MIC did not result in increased killing of biofilm cells.
To investigate the mode of action of this new class of peptides, we used microarray analysis to
compare the transcript profiles of planktonic C. albicans cells exposed to ApoEdpL-W (2.5 µM)
for 10 and 30 min and of unexposed cells. GO term analysis of the 176 up-regulated genes and the
225 down-regulated genes highlighted the over-expression of genes encoding amino acid and
peptide transporters. In particular the GAP6, GAP1, CAN1, GNP1, and PTR2 genes were among
the most up-regulated genes at both time points. On the opposite, a significant fraction of the
genes involved in the methionine biosynthesis pathway, were significantly down-regulated 10 min
after exposure even though to a relatively low extent. These transcript profiling data were
confirmed by qRT-PCR. Interestingly, the transcriptional response of ApoEdpL-W-exposed C.
albicans cells appeared to differ from that exhibited in response to other antifungals including
histatin-5, a well characterized antifungal peptide, thus suggesting that ApoEdpL-W may have a
distinct mode-of-action. Current experiments are aimed at evaluating the fate of ApoEdpL-W in
C. albicans cells using fluorescent derivatives.




                                                147
P98B
Development of a universal system for fungal species identification and SNP typing via on-
chip minisequencing
Michaela K. Mai1, Manuela Gfell2, Nicole C. Hauser2, Bettina Rohde3, Christopher Bauser3,
Selene Ferrari4, Alix Coste4, Dominique Sanglard4, Oliver Bader5, Michael Weig5, Uwe Gross5,
Emilia Mellado6 and Steffen Rupp2
1 MBT, IGVT , Universität Stuttgart, Nobelstr. 12, Stuttgart 70569, GERMANY, Phone: +49 711
970 4171, FAX: +49 711 970 4200, e-mail: michaela.mai@igb.fhg.de
2 Fraunhofer Institut für Grenzflächen- und Bioverfahrenstechnik (IGB), Department of Molecular
Biotechnology, Nobelstr. 12, 70569 Stuttgart, Germany
3 GATC Biotech AG, Jakob-Stadler-Platz 7, 78467 Konstanz, Germany
4 Centre Hospitalier Universitaire Vaudois, Institut de Microbiologie, Rue du Bugnon 48, 1011
Lausanne, Switzerland
5 University Medical Center Göttingen, Georg-August-University, Institute of Medical
Microbiology, Kreuzbergring 57, 37075 Göttingen, Germany
6 Instituto de Salud Carlos III, Servicio de Micologia Centro Nacional de Microbiologia,
Carreterra Majadahonda-Pozuelo km2, 28.220 Majadahonda, Madrid, Spain

Fungal infections are a predominant clinical problem, especially in intensive care units. In
particular for patients with a defective immune system, fungal infections are associated with high
mortality rates. Nevertheless, a fast and well directed medication may improve patient outcome
significantly. A further problem is the increasing resistance against the leading antimycotics due to
the rise of inherently resistant fungal species or during long term treatment. The resistance
mechanisms of fungal pathogens are often based on single nucleotide polymorphisms (SNPs) in
genes regulating the expression of pumps extruding the drug outside the fungal cell or encoding
for the target of the antimycotica. Since development of resistance is not predictable, constant
resistance monitoring is necessary to enable adequate patient medication. In the present study we
developed a system for the highly parallel detection of fungal species and their SNPs associated
with azole resistance using an on-chip minisequencing technology.
Minisequencing allows parallel analysis of SNPs both in homo- and heterozygous strains and
offers a good platform in terms of species identification. For the minisequencing reaction the
spotted probes are hybridized with PCR products from clinical samples and synthetic control
probes. In the enzymatic reaction, different fluorescently labelled dideoxynucleotides and a
thermo stable sequenase are used for the specific extension of the probes with the perfectly
matching nucleotide.
Based on this system, we developed a prototype chip with 15 species specific probes for Candida
albicans, C. glabrata and Aspergillus fumigatus based on ITS or 18s rRNA sequences as well as
SNP probes for erg11, tac1 and mrr1 SNPs of C. albicans from our existing SNP chip. Mutations
in these genes are central for causing resistance. Furthermore, four control probes used to confirm
the correct incorporation of the fluorescently labelled didesoxynucleotides have been developed to
enable normalisation, which is essential for correct identification of heterozygosity. The chip has
been successfully validated with synthetic templates and with defined PCR products from clinical
isolates. In the future, the chip will be expanded by further resistance associated SNPs and by on-
chip minisequencing based species identification probes. This work has been financially supported
by the EURESFUN project (EU-FP6-STREP).




                                                148
P99C
Characterization of S. cerevisiae strains lacking the azole target 14 alpha sterol demethylase
(ERG11) and expressing various alleles of A. fumigatus cyp51A
Laura Alcazar Fuoli1, Emilia Mellado2 and Dominique Sanglard3
1 Department of Microbiology, Imperial College London, South Kensington Campus, London
SW7, United Kingdom, Phone: 004420 7594 5293, FAX: 00442075943076, e-mail: l.alcazar-
fuoli@imperial.ac.uk, Web: http://www3.imperial.ac.uk
2 Servicio de Micologia, ISCIII, Madrid, Spain
3 Institute of Microbiology, University of Lausanne and University Hospital Center, Lausanne,
Switzerland

Background
Resistant strains of Aspergillus fumigatus to azole drugs have been recently detected and the
underlying molecular mechanisms of resistance characterized. Point mutations in cyp51A gene
have been proved to be related to azole resistance in A. fumigatus strains and different resistance
profiles can be attributed depending on the amino acid change. The aim of this work was the
heterologous expression of A. fumigatus cyp51A genes in the yeast S. cerevisiae to assess the
contribution of each independent mutation (G54E, G54V, G54R, G54W, M220V, M220K,
M220T, M220I).
Methods and material
The functional complementation was performed by conditional expression of the yeast ERG11
gene with a tetracycline regulatable system and induced expression of Cyp51A cDNAs from A.
fumigatus. A yeast mutant lacking major efflux transporters (PDR5) was first constructed. This
strain was transformed with the cyp51A cDNAs previously amplified by PCR and the
pYESCT/CT plasmid. Transformants were screened in a selective media containing YNB ura-,
and then tested with doxycycline plus galactose. The positives clones were analyzed by western-
blot and the amplification and sequencing of the cyp51A genes was done. Susceptibility testing to
azoles by E-test was performed usine a selective media (YNB ura-), galactose and doxycycline.
Results
A total of sixty mutants were doxycycline positives and thirty four mutants were verified for their
Cyp51A proteins expression. At least two mutants for each cyp51A mutation were chosen for
azole susceptibility testing.
Conclusions
(i) The lack of S. cerevisiae Erg11 is efficiently complemented by expression of any Aspergillus
cyp51A variant; (ii) there is a marked difference between the MICs values of those clones with
cyp51A from a resistant strain compared with the wild type cyp51A (statistically significant P
value <0.05) for itraconazole and posaconazole; (iii) all clones with a mutated cyp51A copy were
resistant to fluconazole compared with the wild type; (iv) clones with the G54W and M220K
genetic background showed the highest MICs values to itraconazole and posaconazole; (v) the
precise impact of each substitution remains to be fully elucidated. Some differences on the
susceptibilities between clones with the same background were observed. These differences could
be due to some extra mutations that were found in some clones although we can not rule out an
increased expression depending on the number of copies of the plasmid.




                                               149
P100A
COMPARATIVE analysis of the cell wall proteome of Candida albicans grown at acidic
and neutral pH
Grazyna J. Sosinska, Alice Sorgo, Clemens Heilmann, Piet W.J. De Groot, Leo De Koning,
Henk L. Dekkers, Chris De Koster, Stanley Brul and Frans M. Klis
Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht
166, Amsterdam 1018 WV, NETHERLANDS, Phone: +31 (0) 6 5757 4190, FAX: +31 (0)
20525 7924, e-mail: a.g.sorgo@uva.nl

The cell wall of Candida albicans contains at any time more than twenty different covalently
linked mannoproteins varying widely in function (1,2,3). Their precise location in the wall also
varies. To mimic mucosal infections, we developed an in vitro system based on the use of low-
agarose plates containing mucin as the sole nitrogen source. Under these conditions, biomats
were formed that extended with a constant radial growth rate of about 30 micrometer/h. At pH
4, which is representative for the vaginal pH, the cells largely grew as yeast and pseudohyphal
cells, and invasive growth was very limited, whereas at pH 7, which is representative for oral
infections, the cells rapidly invaded the agarose layer. Quantification of the cell wall
proteomes of pH 4- and pH 7-grown biomats was realized by mixing the cell cultures grown
under the two conditions with a 15N metabolically labeled reference cell culture, followed by
LC-FTMS mass spectrometric 14N/15N peptide ratio measurements in the tryptic lysates. The
identification and quantification of 24 cell wall proteins showed that the cell wall proteome of
C. albicans is highly dynamic. This was reflected in the strong up-regulation at pH 7 of three
adhesion proteins (Als1, Als3, and Hwp1), an iron-acquisition-protein (Rbt5), a defense
protein (the superoxide dismutase Sod5), two proteins involved in cell wall formation (Phr1
and Sim1) and two cell wall proteins with unknown function (Hyr1 and Ihd1/Pga36). The
proteome quantification results were consistent with immunological analysis and showed
strong correlation with transcript profiling data from the literature. Our results show that the
switch from an acidic pH and yeast growth to neutral pH and hyphal, invasive growth is
accompanied by the incorporation of a different set of cell wall proteins. We propose that these
proteins prepare the cells for the new environmental conditions.

A.S., C.H, and F.M.K acknowledge the financial support by the EU Program FP7-214004-2
FINSysB

(1) Castillo, L., et al., (2008) Proteomics 8, 3871
(2) De Groot, P., et al., (2004) Eukaryot. Cell 3, 955
(3) Sosinska, G., et al., (2008) Microbiol. 154, 510




                                             150
P101B
Candida, Aspergillus and the embryonated hen's egg: Reviving an old alternative
infection model to study fungal pathogenesis
Ilse D. Jacobsen, Melanie Skibbe and Bernhard Hube
Microbial Pathogenicity Mechanisms, Hans Knoell Institute, Beutenbergstraße 11a, Jena
07745, GERMANY, Phone: +49 (0) 3641 532 1223, FAX: +49 (0) 3641 532 0810, e-mail:
ilse.jacobsen@hki-jena.de, Web: http://www.hki-jena.de/index.php

In vivo screening of mutants is an essential tool to study pathogenesis. For fungal pathogens,
murine models are considered to be the gold standard and are the most widely used in vivo
infection model. However, ethical considerations, legal issues, availability of facilities and
costs restrict screening of large numbers of mutants in mice. Thus, alternative infection models
based on cell cultures or invertebrates are used. While cell cultures can only mimic certain
aspects of host-microbe interactions, invertebrate hosts and their immune systems may differ
significantly from mammalian models.
To bridge the gap between invertebrate models and mice, we developed a highly reproducible
alternative infection model for Aspergillus fumigatus and Candida albicans using
embryonated eggs infected on the chorio-allantoic membrane (CAM). This model allows
quantification of killing potentials (survival curves), evaluation of immune responses
(cytokine expression patterns) and histological analysis of infection.
A. fumigatus is the predominant fungal pathogen of birds and embryonated eggs are highly
susceptible to infection in a dose-dependent manner. Five laboratory and four mutant strains of
A. fumigatus, fully virulent, partially attenuated or fully attenuated in the mouse model,
showed comparable virulence potential in both systemic mouse infection models and the egg
model. A. fumigatus invaded the CAM and it’s blood vessels causing bleeding and thrombosis
consistent with pathology in the murine lung.
The embryos’ susceptibility to infection with C. albicans decreased with age from highest
susceptibility on day 8 to complete resistance on day 12, probably reflecting maturation of the
immune system. Sixteen C. albicans mutant strains were tested in 10 days old embryos and
showed similar levels of attenuation as in systemic murine models. Histology of lesions
revealed rapid hyphal formation, tissue invasion and recruitment of host immune cells,
reflected by an increase in proinflammatory cytokine expression.
We propose that embryonated eggs can be used as an alternative infection model. Comparably
low costs and ease of handling allow screening of high numbers of mutants to pre-select
strains with virulence defects for further analysis in murine models. As an alternative model it
could decrease the number of mice needed for infection experiments and may allow
researchers without direct access to animal facilities to perform in vivo virulence studies.




                                            151
P102C
Cytokine signaling regulates the outcome of intracellular macrophage parasitism by
Cryptococcus neoformans
Kerstin Voelz and Robin C. May
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United
Kingdom, Phone: +44 (0)121 41 45420, FAX: +44 (0)121 41 45925, e-mail:
kxv468@bham.ac.uk, Web: http://www.biosciences.bham.ac.uk/labs/may/Home.html

The facultative pathogenic yeast Cryptococcus neoformans and C. gattii commonly cause
severe infections of the central nervous system in patients with impaired immunity, such as
HIV-positive individuals, and also increasingly in immunocompetent individuals. Following
inhalation, Cryptococcus is phagocytosed by alveolar macrophages but, unlike many other
pathogens that are killed by macrophages, Cryptococcus can survive and proliferate within
these infected host cells. Moreover, Cryptococcus is able to escape into the extracellular
environment via a recently characterized non-lytic mechanism ('expulsion') and can be
transferred directly from one macrophage to another (lateral transfer). Thus, macrophages have
been proposed as a trafficking vehicle for the yeast to survive and disseminate within the host.
An improved understanding of the interaction between macrophages and Cryptococcus is
therefore critical for the development of effective therapies.

Although it is well established that the host's cytokine profile dramatically affects the outcome
of cryptococcal infections, the molecular basis for this effect is unclear. Here, we report a
systematic analysis of the influence of Th1, Th17 and Th2 cytokines on the outcome of the
interaction between macrophages and cryptococci. We show that Th1 and Th17 cytokines
activate, whereas Th2 cytokines inhibit macrophage functions. The Th1 cytokines IFN-
gamma, TNF-alpha and the Th17 cytokine IL-17 enhanced yeast cell uptake by macrophages
although intracellular proliferation and cryptococcal expulsion rate were not significantly
altered. Interestingly, however, whilst Cryptococcus phagocytosis was not changed when
treated with the Th2 cytokines IL-4 or IL-13, these cytokines significantly increased
intracellular yeast proliferation whilst significantly reducing the occurrence of pathogen
expulsion.

In conclusion, enhanced Th2 cytokine levels seem to result in less effective control of the
yeast by macrophages and thus might favor cryptococcal survival and dissemination leading to
fatal infections of the central nervous system. These results provide a mechanistic explanation
for the observed poor prognosis of Th2 cytokine profile (e.g. in HIV patients) in cryptococcal
disease and therefore, help to define alternative strategies to improve cryptococcosis treatment
by highlighting the potential of cytokine-based therapies.




                                            152
P103A
Multiple roles of Candida albicans-derived cell wall components in human keratinocytes -
Activation of immune response and induction of apoptosis
Jeanette Wagener1, Günther Weindl2, Piet W. de Groot3, Albert de Boer4, Michael Weig4 and
Martin Schaller1
1 Dermatology, University Tübingen, Liebermeisterstrasse 25, Tübingen 72076, Germany,
Phone: +49 (0) 7071 29 86864, FAX: +49 (0) 7071 29 4405, e-mail:
jeanette.wagener@med.uni-tuebingen.de
2 Institute of Pharmacy, Free University of Berlin, Germany
3 Swammerdam Institut of Life Sciences, University of Amsterdam, Netherlands
4 Department of Medical Microbiology, University of Göttingen, Germany

Rapid immune response in Candida infections is mediated by a number of innate recognition
molecules known as pattern recognition receptors (PRRs). PRRs recognize conserved motifs
called pathogen-associated molecular patterns (PAMPs), which represent broad groups of
microbial pathogens or components. The signalling pathways trigger subsequent inflammatory
responses which are crucial for successful host defence against pathogens. Fungal cell wall
components such as beta-glucan and mannoproteins have been shown to stimulate the innate
immune response in myeloid cells in a toll-like receptor-dependent manner, particularly
through TLR2 and TLR4. However, Candida albicans cell wall components that specifically
induce TLR responses in keratinocytes have not yet been investigated in detail.
In our studies we first examined the effect of different cell wall extractions from C. albicans
on TLR gene expression and found an increase of TLR4 and a slight increase of TLR10,
accompanied with an induction of GM-CSF and IL-8 levels, analyzed by quantitative RT-PCR
and ELISA. However, the different cell wall extractions showed no major differences in the
TLR expression pattern and cytokine release.
Surprisingly, stimulated keratinocytes showed a strong growth inhibition after 24h of
treatment with the cell wall components. Analysis by proliferation assays resulted in nearly
90% resting cells. This observed growth inhibition is caused by a strong accumulation of the
cell cycle inhibitor p27Kip1 inside the nucleus. More detailed analysis showed that the cell
cycle inhibition resulted in an increase of apoptotic cells up to 30% after 72h.In EMSA studies
we observed a decreased activated form of the common transcription factor NF-kappaB
between 6h to 12h of stimulation, but found increased levels of active caspase-3.
In conclusion, our results indicate that distinct pattern recognition receptors together trigger
the innate immunity in human keratinocytes by recognizing different structures of C. albicans.
Furthermore, our results demonstrate the diversity of signalling pathways mediated by fungal
cell wall components. Triggering innate immune responses result in the secretion of pro-
inflammatory mediators which is accompanied by growth inhibition and subsequent induction
of apoptosis.




                                            153
P104B
Identification and characterization of a complete carnitine biosynthesis pathway in
Candida albicans
Karin Strijbis, Carlo van Roermund, Guy Hardy, Janny van den Burg, Karien Bloem, Jolanda
de Haan, Naomi van Vlies, Ronald Wanders, Fred Faz and Ben Distel
Medical Biochemistry, AMC, Meibergdreef 15, Amsterdam 1105 AZ, Netherlands, Phone:
+31-20-5665127, FAX: +31-20-6915519, e-mail: b.distel@amc.uva.nl

Carnitine is an essential metabolite that enables intracellular transport of fatty acids and acetyl
units. We have previously shown that the human fungal pathogen Candida albicans relies
exclusively on carnitine-mediated transport of acetyl units while growing on non-fermentable
carbon sources such as fatty acids, acetate or ethanol (1). We now show that C. albicans can
synthesize carnitine de novo and identify the four genes of the pathway. Null mutants of
orf19.4316 (trimethyllysine dioxygenase), orf19.6306 (trimethylaminobutyraldehyde
dehydrogenase) and orf19.7131 (butyrobetaine dioxygenase) lacked their respective enzymatic
activities and were unable to utilize fatty acids, acetate or ethanol as sole carbon source, in
accordance with the strict requirement for carnitine-mediated transport under these growth
conditions. The second enzyme of carnitine biosynthesis, hydroxy-trimethyllysine aldolase, is
encoded by orf19.6305, a member of the threonine aldolase (TA) family in C. albicans. A
strain lacking orf19.6305 showed strongly reduced growth on fatty acids and was unable to
utilize either acetate or ethanol, but TA activity was unaffected. Growth of the null mutants on
non-fermentable carbon sources is only restored by carnitine biosynthesis intermediates after
the predicted enzymatic block in the pathway, providing independent evidence for a specific
defect in carnitine biosynthesis for each of the mutants. In conclusion, we have genetically
characterized a complete carnitine biosynthesis pathway in C. albicans and show that a TA
family member is mainly involved in the aldolytic cleavage of hydroxy-trimethyllysine in
vivo. Furthermore, we show that the availability of the substrate of the carnitine biosynthesis
pathway, 6-N-trimethyllysine (TML) and thereby carnitine itself is rate limiting during growth
on non-fermentable carbon sources underscoring the importance of the pathway for alternative
carbon source utilization by this pathogenic fungus.

1. Strijbis, K., et al. (2008), Eukaryot. Cell 7, 610




                                              154
P105C
Investigation of arginase in human pathogenic fungus Schizophyllum commune
Arsen Gasparyan1, Armine Nikoyan1, Siranush Nanagulyan2 and Nikolay Avtandilyan1
1 Biochemistry, Yerevan State University, A. Manoogian str. 1, Yerevan 0025, Armenia,
Phone: (+374 99) 238686, FAX: (+374 10) 55-46-41, e-mail: gasparyan.arsen@yahoo.com
2 Department of Botany, Yerevan State University, A. Manoogian str. 1, 0025, Yerevan,
Armenia

The homobasidiomycetes Schizophyllum commune is the rare higher fungi associated with
human infections. Infections reported for Schizophyllum commune include
basidioneuromycosis, allergic fungal sinusitis, ulcerative lesions of the hard palate, fungal ball
of the lung, allergic bronchopulmonary mycosis, mucoid impaction of the bronchi, and brain
abscess (Buzina et al., 2001). The aim of our researches was investigating of arginase activity
and properties in human pathogenic fungus Schizophyllum commune. It is commonly known
that arginase activity limits nitric oxide (NO) production by inhibition inducible NO synthase
(iNOS), by limiting L-arginine availability. It was shown that arginase allows several human
pathogenic organisms (Helicobacter pylori, Paracoccidioides brasiliensis) to avoid the
immune response by regulating eukaryotic NO production (Gobert et al., 2001; Gonzalez et
al., 2000). Our investigation showed high arginiase activity in mycelial culture of
Schizophyllum commune. The study of subcellular localization revealed presence of two
different isoforms of arginase - arginase I (cytosolic enzyme) and arginase II (mitochondrial).
In contrast to arginase I, arginase II constitutes the majority of total activity. The fungal
arginase exhibited pH optimum (9.0 -10.0) and required Mn2+ ions for activity, other bivalent
ions (Co2+, Ni2+, Cd2+) have inhibitory effect. L-arginine was added to the growth medium
to explore its inducing activity. The arginase expression was induced in response to the
intracellular accumulation of L-arginine. Acknowledging this, exploration of the role and
properties of arginase in the pathogenesis of Schizophyllum commune -related disease should
be considered important.

1. Buzina W et al., (2001), J. Clin. Microbiol., 39(7), 2391
2. Gobert A et al., (2001), PNAS, vol. 98, 13844
3. Gonzalez A et al., (2000) Infect Immun., 68(5), 2546




                                             155
P106A
Stage specific gene expression profiling during initiation of invasive aspergillosis
Timothy Cairns1, Dan Chen2, Andrew McDonagh1, William Nierman2, Natalia Fedorova2 and
Elaine Bignell1
1 Department of Microbiology, Imperial College London, Armstrong Road, London SW7
2AZ, England, Phone: 0044 02075945293, FAX: 0044 02075943095, e-mail:
t.cairns07@imperial.ac.uk, Web: http://www3.imperial.ac.uk/cmmi
2 The J. Craig Venter Institute, Rockville, Maryland, United States of America

Early time points of Aspergillus fumigatus infection present the ideal stage for effective
antifungal therapy and disease diagnosis. Sequencing of the A. fumigatus genome has enabled
the development of powerful molecular tools for the analysis of disease initiation. Whole
genome microarrays have previously been constructed to investigate transcriptional regulation
throughout spore germination, antifungal treatment and temperature stress in vitro. However,
of considerable interest is transcriptional regulation during early mammalian infection. We
have developed a methodology for stage-specific gene expression analysis of both host and
pathogen transcriptomes throughout a time-course of murine aspergillosis. Groups of 8
immunosuppressed male CD1 mice were intranasally inoculated with A. fumigatus spores.
Pathogen RNA from invasive germlings was isolated by bronchoalveolar lavage sampling at 4,
8 and 14 hours post infection. Fungal RNA was pooled within groups providing sufficient
concentrations for amplification and microarray analysis. Murine lung RNA was also sampled
at the respective time points for analysis by SuperArray qRT-PCR. We are currently using this
methodology to probe the role of fungal secondary metabolites during invasive infection by
comparing transcriptional regulation throughout murine infection between the Af293 clinical
isolate and a laeA deletetion strain, which misregulates gene expression at multiple secondary
metabolite gene clusters and is avirulent in the murine model. We predict that secondary
metabolite clusters misregulated during infection in the delta laeA strain will identify gene
clusters essential for virulence. The development of this methodology will enable future
investigations of host and pathogen transcriptomes throughout early murine infection for
members of the Aspergillus genus. We predict this may identify much needed targets for
diagnostic design and therapeutic benefit.

McDonagh, A, et al. (2008) PLOS Pathogen, 4.




                                           156
P107B
C. albicans interaction with epithelial cells induces apoptosis and necrosis
Coralie L'Ollivier1, Bernhard Hube2, Alain Bonnin1, Néijia Sassi3 and Frédéric Dalle1
1 laboratoire LIMA (EA562), université de médecine Dijon France, 2 bd du Maréchal de
Lattre de Tassigny, Dijon 21000, France, Phone: +33 (0)3 8029 3780, FAX: +33 (0)3 8029
3627, e-mail: coralie.lollivier@chu-dijon.fr
2 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product
Research, Infection Biology – Hans Knoell Institute Jena (HKI), Jena, Germany and Friedrich
Schiller University, Jena, Germany
3 UMR 866 NO et Cancer Dijon, France

C. albicans is an opportunistic fungus responsible for a wide range of diseases, ranging from
superficial to systemic infections. When causing invasive diseases, C. albicans has to cross
tissue barriers, invading normally non-phagocytic host cells such as oral or intestinal cells.
We have previously confirmed that C. albicans adhered to and invaded more strongly oral
cells. Moreover, C. albicans was able to penetrate oral cells by two distinct cellular
mechanisms: (i) cellular endocytosis and (ii) an active penetration mechanism. Surprisingly, in
interactions with enterocytes, only the active penetration process of the fungus was observed.
Finally, as a consequence of the invading process, cellular damage was directly proportional to
the level of tissue invasion and was detected earlier in oral cells than in enterocytes.
Cell death is induced by a number of pathogens and can include an upregulation or
dowregulation of different host cell death pathways, i.e. oxidative stress, apoptosis and
necrosis. Currently, there is few knowledge about i) the mechanisms of cell death involved
during the interaction between C. albicans and epithelial cells and ii) the relationships between
these different events. To address these questions, apoptosis and necrosis were monitored
using in vitro models of interaction of C. albicans SC5314 with (i) an oral cell line (TR-146)
and (ii) an enterocytic cell line (Caco-2).
In both epithelial cell lines, apoptotic cells co-localized with invading C. albicans cells.
Moreover, invasiveness of the fungus correlated with the magnitude of apoptosis that was
higher in oral cells than in intestinal cells. Finally, necrosis was detectable after 3 h and 6 h of
infection in oral and intestinal cells respectively. Our data suggest that the dynamics of the «
apoptosis – necrosis » sequence is dependent on the epithelial cell type. Whether apoptosis
induces necrosis and how cellular endocytosis by oral cells and active penetration of the
fungus contribute to these processes are currently under investigation, using inhibitors of
endocytosis (Cytochalasin D) and apoptosis (z-VAD-fmk). In a second step, oxidative stress
will be monitored and replaced in relation with apoptotic and/or necrotic processes.




                                              157
P108C
TLR2/MyD88-dependent and -independent activation of mast cell IgE responses by the
skin commensal yeast Malassezia sympodialis
Christine Selander, Camilla Engblom, Gunnar Nilsson, Carolina Lunderius Andersson and
Annika Scheynius
Dept of Medicine Solna, Karolinska Institutet, L2:04, Stockholm 171 77, Sweden, Phone: +46
8 5177 5934, FAX: +46 8 335724, e-mail: annika.scheynius@ki.se

Atopic eczema (AE) is a chronic inflammatory skin disease. Approximately 50% of adult AE
patients have allergen-specific IgE-reactivity to the skin commensal yeast Malassezia spp. Due
to the ruptured skin barrier in AE it is likely that Malassezia can come into contact with mast
cells, which are known to be involved in AE. We therefore hypothesized that Malassezia spp
can activate mast cells. Bone marrow-derived mast cells (BMMCs) were generated from wild
type (Wt), TLR2, TLR4 and MyD88 gene deleted mice and co-cultured with M. sympodialis
extract. We recorded that M. sympodialis induced release of cysteinyl leukotrienes in a dose-
dependent manner in non-sensitized and IgE-anti-trinitrophenyl (TNP)-sensitized BMMCs,
respectively, with three times higher levels in the latter type of cells. IgE-sensitized BMMCs
also responded by degranulation as assessed by release of beta-hexosaminidase, increased
MCP-1 production through a MyD88-independent pathway and activated phosphorylation of
the MAPK ERK 1/2. Furthermore, M. sympodialis enhanced the degranulation of IgE-receptor
cross-linked Wt BMMCs and altered the IL-6 release dose-dependently. This degranulation
was independent of TLR2, TLR4 and MyD88, whereas the IL-6 production was dependent on
the TLR2/MyD88 pathway and MAPK signaling. In conclusion, M. sympodialis extract can
activate non-sensitized and IgE-sensitized mast cells to release inflammatory mediators, to
enhance the IgE-mediated degranulation of mast cells, to modulate MAPK activation and by
signaling through the TLR2/MyD88 pathway to modify the IL-6 production of IgE-receptor
cross-linked mast cells. Collectively, these findings indicate that M. sympodialis can activate
mast cells and might thus exacerbate the inflammatory response in AE.




                                           158
P109A
Comparative transcriptional profiling of Candida albicans identifies novel infection-
associated genes
Duncan Wilson, Francois Mayer and Bernhard Hube
Microbial Pathogenicity Mechanisms, Hans Knoell Institute, Beutenbergstrasse, 11a, Jena
07745, Germany, Phone: +49(0)532 12 13, FAX: +49(0)3641 532 08 10, e-mail:
Duncan.Wilson@hki-jena.de, Web: http://www.hki-jena.de/index.php

We have performed in vitro and in vivo transcriptional profiling of Candida albicans using
models of oral, liver and blood infections. A large number of genes transcriptionally up-
regulated during infection encoded proteins of unknown function and a number of these lacked
orthologues in other fungal species. We reasoned that these infection-associated genes (the C.
albicans “infectome”) constitute potential pathogenicity factors involved in the initiation and
persistence of infection. Furthermore, differential expression patterns during different types of
infection indicated that certain gene products would be of pathogenic significance in a niche-
or stage-specific manner. To date we have deleted over 40 infection-associated genes and
begun to analyse their role in pathogenesis: the phenotypes of selected mutants during host-
interactions will be presented. For example, OCS2 (oral infection-induced cell surface) is up-
regulated during both in vitro and in vivo oral infections and encodes a protein with eight
putative transmembrane helices. Deletion of OCS2 prohibited filamentation under embedded
conditions, but not in response to other hyphal-induction media, and resulted in significantly
reduced damage of human oral epithelial cells. BIS1 (blood-induced stress protein) contains a
small heat shock domain and was shown to be required for both growth at elevated
temperature (42°C) and host cell damage, demonstrating a novel link between thermal
tolerance and pathogenicity during oral infection. MOP1 (mid-phase oral infection protein)
encodes a protein with no conserved structural/functional domains. Deletion of this gene
resulted in a stage-specific defect in oral epithelial cell infection: damage caused between 8 h
and 15 h post-inoculation was arrested upon deletion of MOP1. This example reinforces our
concept that specific factors are required for C. albicans infection depending not only on the
anatomical niche, but the specific stage of infection. We are currently analysing the behaviour
of our mutant set in a wider range of infection models, such as during interactions with
immune cells. As an additional infection model, we have developed a circulatory system to
analyse C. albicans-endothelial cell-interactions under conditions of physiological flow. We
provide evidence that both the yeast to hyphal transition and the appropriate expression of cell
surface proteins are required for fungal adhesion under this condition.




                                            159
P110B
Neutrophil extracellular traps and Aspergillus fumigatus
Sandra Wolke1, Franziska Lessing1, Mike Hasenberg2, Alexander Gehrke1, Olaf Kniemeyer1,
Matthias Gunzer2 and Axel Brakhage1
1 Molecular and Applied Microbiology, Hans Knoell Institute, Beutenbergstrasse 11a, Jena
07743, GERMANY, Phone: +49 (0)3641 532 1094, FAX: +49 (0)3461 532 0803, e-mail:
Sandra.Wolke@hki-jena.de, Web: www.hki-jena.de
2 Institute for Molecular and Clinical Immunology, Medical Faculty, Otto-von-Guericke-
University, Leipziger Str 44, Magdeburg 39120, GERMANY

With the increasing number of immunocompromised individuals Aspergillus fumigatus has
become the most important opportunistic fungal pathgen. Conidia as the infectious agent
infiltrate the lungs and get in contact with the human immune system. The first line of defense
is represented by alveolar macrophages and neutrophil granulocytes. From Candida albicans it
is known that neutrophils are able to attack the pathogen by beneficial suicide (Brinkmann and
Zychlinsky, 2007). In this ROI dependent mechanism the neutrophils release DNA filaments
covered with histones and granule proteins. These sticky filaments are known as neutrophil
extracellular traps (NETs). Steinberg and Grienstein (2007) named this process NETosis. We
coincubated A. fumigatus germlings with human neutrophils for up to three hours and took
samples for CLSM and SEM analysis. NET like structures were clearly visible. Furthermore,
we filmed the direct process of NETosis. To analyse the role of NET formation in killing of A.
fumigatus during coincubation we used an XTT assay. Moreover, to investigate the
dependency of NET formation on the induction of an oxidative burst we added the NADPH-
oxidase inhibitor DPI and the ROI scavenger glutathione. Inhibition of ROI production
apparently led to reduced NET formation. Taken together, we showed that neutrophils form
NETs after contact with A. fumigatus mycelium. Furthermore, NET formation was dependent
on ROI formation. We propose that NETs have the function to agglutinate A. fumigatus
hyphae, to constrain the infection, and to recruit additional immune cells.




                                           160
P111C
Genetic analysis of C. albicans dissemination & colonization of host niches
Lanay Tierney, Olivia Majer, Christelle Bourgeois and Karl Kuchler
Department of Medical Biochemistry, Max F. Perutz Laboratories, Dr. Bohr-Gasse 9/2,
Vienna 1030, Austria, Phone: +43 (0)1 4277 6181 2, FAX: +43 (0)1 4277 9618, e-mail:
Lanay.Tierney@meduniwien.ac.at,                                               Web:
http://www.meduniwien.ac.at/medbch/MolGen/kuchler/

Common commensal pathogens of the GI tract and oral cavity of healthy individuals, Candida
spp and particularly C. albicans (Ca), can cause fatal multi-organ infections in the
immunocompromised. Each organ or niche presents a different microenvironmental challenge
that the pathogen must be able to adapt for successful colonization. The unique plasticity of
the Ca genome makes it adaptable to almost all organs within the host. However, the speed,
the extent of this adaptation and how it takes place is not well understood. We aim to study
this commensal pathogen and characterize the complex niche adaptation, in molecular terms,
in order to device strategies to limit dissemination in, or colonization of, mammalian hosts by
Ca. To study Ca genetic adaptation in a host, we will look at Ca infections as both virulent
dissemination and in the avirulent commensal state in characteristically distinct niches. We
shall ask how distinct host niches change the Ca genome, and how niches drive
microenvironmental adaptation. Quantitative in vitro and in vivo transcriptome analysis of
pathogens in the host will be performed for each niche at different stages of infection using
deep sequencing. Using comparative genomics and bioinformatics, it may be possible to
identify niche-specific and niche-independent virulence genes of Ca and their expression
pattern within a host. With virulence patterns emerging, they can then be compared to the
commensal situation to analyze the opposing extreme. Although not normally a murine
commensal, colonization of mice by Ca can be induced though physical or chemical
manipulation. Similarly, after exposure of the pathogen to the host immune system, we shall
ask whether or not Ca acquires a “memory” of its niche in such a way that they will
preferentially localize to their “home” niche upon reinoculation in mice. To address this
question, we will follow fluorescently labeled clinical isolates and mutant Ca strains during
infections using in vivo real time imaging systems. Together these questions aim to identify
the genetic strategies the pathogen exploits for colonization of diverse host niches. By
understanding how the pathogen adapts, it is possible to deduce therapeutic interventions and
limit its dissemination in vivo.

The work is supported by the Christian Doppler Research Society, a OeAW-DOCff-FORTE
PhD Studentship to OM, and a VBC PhD Programme fellowship to LT.




                                           161
P112A
Molecular analysis of the response of cultured A549 lung cells to A. fumigatus infection
Haim Ben-Zvi (Sharon) and Nir Osherov
Department of Human Microbiology and Immunology, Tel Aviv University, Haim Levanon,
Ramat-Aviv, Tel Aviv 69978, ISRAEL, Phone: +97236409946, FAX: +97236409160, e-mail:
haimpossible@gmail.com

Aspergillus fumigatus is the most prevalent airborne fungal pathogen which causes fatal
invasive aspergillosis in immunocompromised patients. Invasive pulmonary aspergillosis
(IPA) is caused by inhalation of A. fumigatus spores and growth of the fungus inside the lungs,
often spreading from the initial site of infection in the lungs to attack various organs in the
body. Our main goal is to better understand the mechanisms controlling damage in infected
lung alveolar epithelial cells. We assumed that lung alveolar cells infected with A. fumigatus
undergo a specific and pre-programmed molecular response. In order to reveal the processes
which take place during aspergilosis pathogenesis, we examine key elements which undergo
phosphorylation and transcriptional modifications. Our results demonstrate that A549 lung
cells respond differentially to infection with A. fumigatus conidia or culture filtrate. We
propose that during early conidial infection, infected cells mount a vigorous protective
response characterized by up regulation of cytokines, signaling pathways and transcription
factors. In contrast, during late infection, the accumulation of secreted culture filtrate elicits a
marked inhibition of cellular metabolism, characterized by the shutdown of amino acid
metabolism, reduced activity of transcription factors and reduction of protective responses. We
evaluated the effect of infection on structural (cytoskeletal reorganization, cell peeling, We
had also created and tested a protease deficient strain, which is completely devoid of
proteolytic activity, loss of cell viability) and molecular responses of infected A549 cells,
using several inhibitor compounds. These results help clarify the progression of cellular
infection by A. fumigatus at the molecular level, and suggest novel ways to interfere with this
destructive process.




                                              162
P113B
Arthroderma benhamiae uses a dual strategy to evade host complement attack
Susann Schindler, Peter F. Zipfel and Axel A. Brakhage
Infection Biology, Leibniz Institut -Hans Knöll Institut, Beutenbergstrasse 11a, Jena 07745,
Germany, Phone: 036415321168, FAX: 036415320807, e-mail: susann.schindler@hki-
jena.de, Web: www.hki-jena.de

Dermatophytes cause human cutaneous mycosis, which represent a prevalent worldwide health
problem. Immune evasion of dermatophytes is important for virulence and pathogenicity,
therefore it is of interest to understand immune escape strategies of these pathogens.
Arthroderma benhamiae is investigated as a model organism for skin infections. The
complement system forms the first line of immune defense against invading microorganisms
in the human skin. Human complement regulator Factor H mediates degradation of
complement C3b, a major opsonisin for phagocytosis. We demonstrate that Factor H is locally
expressed by human keratinocytes, which is shown by Western Blot analysis of culture
supernatant. Furthermore Factor H binds to the surface of A. benhamiae, as shown by direct
binding assays, immunostaining and ELISA. Pathogen bound Factor H inhibits the deposition
of C3b on the fungal surface. Thus, the dermatophyte avoids opsonization by host complement
C3b and subsequent phagocytosis.
In addition A. benhamiae uses a second independent strategy to block human complement. The
fungus secretes proteases, which can degrade complement components as revealed by
cleavage assays and haemolytic tests. This implies that A. benhamiae protects itself against
complement attack by interfering with the complement activation.
A kinetic study of C3b degradation shows, that it is essential for A. benhamiae survivial to
bind host complement regulator Factor H in the first few minutes of infection to mediate C3b
inactivation. At a later time point secreted fungal proteases take over the role of C3b
degradation. These results suggest, that A. benhamiae uses two subsequently independent
immune escape strategies.




                                          163
P114C
Using tissue engineered oral mucosa to identify innate defence mechanisms against yeast
and hyphal forms of Candida albicans
Nishant Yadev1, Craig Murdoch1, Stephen Saville2, Jose Lopez-Ribot2 and Martin Thornhill1
1 Oral and Maxillofacial Medicine and Surgery, School of Clinical Dentistry, University of
Sheffield, 19 Claremont Crescent, Sheffield S10 2TA, UK, Phone: +44 (0) 114 271 7964,
FAX: +44 (0) 114 271 7863, e-mail: n.yadev@shef.ac.uk
2 Dept. of Biology, Tobin Building, West Campus, UTSA, One UTSA Circle, San Antonio,
Texas, USA

Candida albicans is part of the normal commensal microbial flora of healthy individuals.
However, under certain circumstances C. albicans can undergo morphogenic transformation
from its commensal yeast form to a more pathogenic hyphal form that can cause infection. Of
the infections generated by C. albicans, oropharyngeal candidiasis is the most commonly
encountered. Clinical and experimental studies have shown that local host innate and immune
defence mechanisms of the oral mucosa are important in providing host defence against
infection and maintaining a commensal relationship. However, very little is known about the
mechanisms that are involved.
This study used a genetically modified strain of C. albicans (SSY50B), in which the NRG-1
gene has been placed under the control of a doxycycline (DOX) regulatable promoter. In the
absence of DOX, SSY50B remain in their yeast form but in the presence of DOX they undergo
hyphal transformation. We also used a full thickness mucosal model system to mimic the oral
mucosa in vivo. The infection of this model system with SSY50B, in the presence or absence
of DOX enabled us to study the role of yeast to hyphal transformation in an in vitro model of
oral candidiasis.
Infection of the model system with SSY50B+DOX (hyphal form) showed increased levels of
tissue damage and hyphal invasion over time, as observed by histological examination and
lactate dehydrogenase release, than SSY50B-DOX (yeast form), although the latter form of C.
albicans still caused considerable damage itself. CAF2 the control, wild-type parent strain was
intermediate in its effect. By 48 hours the tissue damage caused by all 3 forms of Candida was
similar. In addition, we quantified the release of cytokines by the oral epithelial cells using
cytokine arrays and ELISA. Over a 48 hour period all 3 forms of C. albicans induced a large
increase in GM-CSF and CXCL8 release. Cytokine release was most rapid with
SSY50B+DOX, least with SSY50B-DOX and CAF2 was intermediate in its effect.
Nonetheless, by 48 hours the level of cytokine release induced by all 3 was similar. In contrast,
SSY50B+DOX, induced a large increase in IL-1B release by 48 hours but SSY50B–DOX did
not. CAF2 was intermediate in its effect.
These data suggest that the hyphal form of C. albicans is more invasive and rapidly destructive
to epithelium, although within our model system, yeast forms were capable of causing similar
levels of tissue damage and cytokine release by 48 hours.




                                            164
P115A
Identification of virulence factors in Histoplasma capsulatum
Alison Coady, Dervla Isaac and Anita Sil
Department of Microbiology and Immunology, University of California, San Francisco, 513
Parnassus Ave, Box 0414, San Francisco CA 94143, USA, Phone: +1-415-502-4810, FAX:
+1-415-476-8201, e-mail: alison.coady@ucsf.edu

Histoplasma capsulatum is a dimorphic fungal pathogen well adapted to survive within the
macrophage. The organism exists in the environment as a filamentous mycelial form that is
easily aerosolized and inhaled by the host. Inside the host, the mycelial form converts into a
pathogenic yeast form. H. capsulatum yeast are able to survive and replicate within host
macrophages, eventually causing cell lysis. In a healthy host, infection by H. capsulatum is
limited by cell-mediated immune responses. However in an immunocompromised host,
infection by H. capsulatum leads to a disseminated and often fatal disease. The molecular
strategies employed by Histoplasma to survive and replicate inside macrophages are not well
understood. Only two virulence factors, Yps3 and Cbp1, have been identified in the H.
capsulatum strain G217B. To identify additional mechanisms contributing to the survival and
virulence of H. capsulatum, our lab employed a high-throughput insertional mutagenesis
screen to identify mutants defective in macrophage lysis. A screen of 14,000 H. capsulatum
insertion mutants identified 47 lysis defective (ldf) mutants. At least two of these mutants
contain disruptions in the CBP1 gene, providing validation that the screen identified genes
required for macrophage colonization and lysis. The remainder of the lysis defective (ldf)
mutants display moderate to severe macrophage lysis defects as measured by a quantitative
cell-lysis assay. We have identified mutants that fail to survive within macrophages, as well as
mutants which grow inside macrophages but are unable to lyse the cell. Using the putative
function of the genes disrupted and preliminary characterization of the lysis defects exhibited
by the mutants, we have selected ten mutants to further characterize. These mutants are
defective in genes which encode potential secreted proteins as well as proteins involved in
transport, metabolism and signaling. We are currently determining which of these mutants
display reduced virulence in a mouse model of histoplasmosis.




                                            165
P116B
Microbial quorum sensing molecules induce multiple damages in human spermatozoa
Claudia Rennemeier2, Johannes Dietl2 and Peter Staib1
1 Fundamental Molecular Biology of Pathogenic fungi, Hans-Knoell-Institute, Beutenbergstr.
11a, Jena 07745, Germany, Phone: +49 (0) 3641 532 1600, FAX: +49 (0) 3641 532 0809, e-
mail: peter.staib@hki-jena.de, Web: www.hki-jena.de
2 Department of Obstetrics and Gynecology, University of Würzburg, Josef-Schneider Str. 4,
97080 Würzburg, Germany

Infertility in men and women is frequently associated with genital contaminations caused by
various microorganisms. The molecular basis of this correlation remains still elusive, and little
attention has been paid on potential direct influences of commensal or uropathogenic microbes
on human gametes. Since many microorganisms are known to release distinct signaling
molecules in substantial amounts, we raised the question whether such molecules can directly
affect human spermatozoa. Here we show that the quorum sensing molecules farnesol and 3-
oxododecanoyl-L-homoserine lactone employed by the opportunistic pathogenic yeast
Candida albicans and the gram negative bacterium Pseudomonas aeruginosa, respectively,
induce multiple damages in human spermatozoa. In detail, a reduction in the motility of
spermatozoa coincided dose-dependently with apoptosis and necrosis at concentrations which
were non-deleterious for dendritic-like immune cells. Moreover, sublethal doses of both
signaling molecules induced premature loss of the acrosome, a cap-like structure of the sperm
head which is essential for fertilization. This work uncovers a new facet in the interaction of
microorganisms with human gametes, and at the same time sheds new light in the
phenomenon of quorum sensing, a microbial communication system which may impact not
only interkingdom signaling and pathogenicity but also host fertility.




                                            166
P117C
Gene expression profiling and gene targeting in human pathogenic dermatophytes
Maria Grumbt1, Christophe Zaugg2, Johann Weber3, Bernard Mignon4, Michel Monod2 and
Peter Staib1
1 Fundamental Molecular Biology of Pathogenic Fungi, Hans Knoell Institute, Beutenbergstr.
11a, Jena 07745, Germany, Phone: +49 3641 532 1247, FAX: +49 3641 532 0809, e-mail:
maria.grumbt@hki-jena.de, Web: www.hki-jena.de
2 Department of Dermatology, University of Lausanne, Av. de Beaumont 29, Lausanne,
Switzerland
3 DNA Array Facility, University of Lausanne, Genopode Building, Lausanne, Switzerland
4 Department of Infectious and Parasitic Diseases, University of Liège, B-43 Sart-Tilman,
Liège, Belgium

Dermatophytes are highly specialized fungi which are the most common agents of superficial
mycoses in humans and animals. The particular ability of these microorganisms to invade and
multiply within keratinized host structures is presumably linked to their secreted keratinolytic
activity, which is therefore a major putative virulence attribute of these fungi. However, the
overall adaptation and transcriptional response of dermatophytes during protein degradation
and/or infection is largely unknown. To address this issue, a Trichophyton rubrum cDNA
microarray was developed and used for the transcriptional analysis of T. rubrum and
Arthroderma benhamiae cells during growth on protein substrates. Since the zoophilic A.
benhamiae causes highly inflammatory dermatophytosis not only in humans but also in
rodents, the gene expression profile in A. benhamiae cells was also monitored during infection
of guinea pigs. In vitro, both T. rubrum and A. benhamiae cells activated a large set of genes
encoding secreted endo- and exoproteases during utilization of soy and keratin. In addition,
other specifically induced factors with potential implication in protein utilization were
identified, e.g. multiple transporters, metabolic enzymes of the glyoxylate cycle, transcription
factors and hypothetical proteins with unknown function. Notably however, the protease gene
expression profile in the fungal cells during infection was significantly different from the
pattern elicited during in vitro growth on keratin, suggesting specific functions of individual
proteases during infection. For functional analysis of putatively virulence associated genes a
transformation system for targeted gene disruption in A. benhamiae was established, and
candidate mutants are currently phenotypically analysed. In conclusion, this first broad in vivo
transcriptional profiling approach in dermatophytes gives new molecular insights into
pathogenicity associated adaptation mechanisms that make these microorganisms the most
successful causitive agents of superficial mycoses.




                                            167
P118A
Interaction of human primary immune cells with Aspergillus fumigatus under the
influence of immunomodulatory agents
Ruth Bauer1, Markus Mezger1, Christian Blockhaus1, Oliver Kurzai2, Hermann Einsele1 and
Juergen Loeffler1
1 Medizinische Klinik und Poliklinik II, Universitaet Wuerzburg, Josef-Schneider Str. 2,
Wuerzburg 97080, Germany, Phone: +49 (0)931 201 36408, FAX: +49 (0)931 201 36409, e-
mail:          bauer_r1@klinik.uni-wuerzburg.de,      Web:         http://www.klinik.uni-
wuerzburg.de/deutsch/home/content.html
2 Institut für Hygiene und Mikrobiologie

Mycophenolate (MPA) and 40-0-[2-Hydroxy-ethyl] rapamycin (RAD) are immunosuppressive
agents. MPA inhibits the proliferation of B- and T-lymphocytes by interfering with the DNA
synthesis, whereas RAD binds to the cytosolic protein FK506 binding protein (FKBP12)
leading to a repression of T-cell activation. Both agents are indicated for the prevention of
solid organ and bone marrow transplant rejection. However, they constitute risk factors for the
development of opportunistic infections, such as invasive pulmonary aspergillosis (IPA),
which is mainly caused by the most common airborne fungal pathogen Aspergillus fumigatus.
Therefore, we investigated the effect of MPA and RAD on polymorphonuclear neutrophils
(PMN), which are essential in the first line of defence against A. fumigatus, and on monocyte-
derived dendritic cells (moDC).
The oxidative burst of PMNs was measured under the influence of MPA or RAD in the
presence of Dichlorofluorescein diacetate. Apoptosis assays were assessed by Annexin V-
FITC and Propidium Iodide staining and subsequent flow cytometry. moDCs were derived
from monocytes upon culture with granulocyte-macrophage colony-stimulating factor and
interleukin-4 in the presence or absence of RAD or MPA. The surface markers CD40, CD80,
CD83 and CD86 as well as ingestion and binding rates to FITC labelled beads were detected
by flow cytometry. Cytokine production was defined by real-time PCR and ELISA assays.
We measured an increased oxidative burst of PMNs confronted with A. fumigatus under the
influence of MPA. Evidence for induction of PMN apoptosis by MPA could not be found. No
effects were observed on fungal viability, when PMNs where primed with RAD.
moDCs showed reduced expression of all surface markers analysed. Moreover pro-
inflammatory cytokine response of moDCs, such as Interleukin-12 and tumor necrosis factor-
alpha, was impaired when RAD or MPA treated cells were used against A. fumigatus.
Furthermore, RAD reduced the phagocytic activity of moDCs.
In conclusion, treatment with MPA or RAD had no inhibitory effects on PMNs. In contrast,
both agents had considerable effects on impairing the function of moDCs, which link innate
and adaptive immune response.




                                           168
P119B
Impact of Type I Interferons on the Cell-Mediated Immunity to Candida Infection
Olivia Majer1, Christelle Bourgeois1, Ingrid Frohner1, Mathias Müller2, Thomas Decker3 and
Karl Kuchler1
1 Medical Biochemistry, Max F. Perutz Laboratories; Medical University of Vienna, Dr.
Bohrgasse 9/2, Vienna 1030, Austria, Phone: +43 1 4277 61812, FAX: +43 1 4277 9618, e-
mail: olivia.majer@meduniwien.ac.at, Web: www.mfpl.ac.at
2 Veterinary University of Vienna
3 Max F. Perutz Laboratories, University of Vienna

The clinical spectrum of diseases caused by Candida spp. ranges from mucocutaneous
infections to systemic, life-threatening diseases in immunocompromised patients. We wish to
identify signaling mechanisms implicated in the host immune response, including a potential
role for type I interferon signaling (IFN). Whereas IFNs are protecting against viral and
bacterial infections, a function in conferring immunity against fungal pathogens has not been
uncovered. Type I IFNs such as IFNb are pleiotropic cytokines with important pro-
inflammatory, as well as immuno-modulatory functions. So-called Th1 and Th17 effector
mechanisms, which are balanced by regulatory T cells, have been associated with the
protection against candidiasis, whereas the development of Th2 immunity is associated with a
non-protective response. T-cell activation and subsequent lineage differentiation requires
specific cytokines, including IFNb, which are released by DCs. Hence, we have used immune
cells such as primary myeloid dendritic cells (mDCs), as well as animal models, to investigate
the role of IFN in fungal infections. Candida spp indeed trigger the release of high IFNb
amounts from mDCs. Importantly, mice lacking the type I IFN receptor IFNAR1 exhibit
hyper-susceptibility to disseminated candidiasis when compared to wild type mice. To further
delineate the impact of type I IFNs on Th-lineage differentiation and thus host immunity, we
have also established a primary splenocyte cell culture system from immunized mice, which is
highly responsive to Candida spp as demonstrated by the production of typical signature
cytokines, including IFNg (Th1), IL-4 (Th2), IL-17 (Th17) and IL-10 (T-reg). Our work
establishes for the first time a function for IFNb in the protection against fungal dissemination,
suggesting a biological role in the clearance of Candida spp. We also suggest that IFNb
mediates the defence against candidiasis by activating T cell immunity. A mDC/T-cell co-
culture system will help us to unravel the molecular signals driving T cell proliferation, and
uncover the mechanisms by which IFNb triggers adaptive cell-mediated immunity against
fungal pathogens.

The work is supported by the Christian Doppler Research Society, the transnational ERA-Net
Pathogenomics project FunPath (FWF-I125-B09), through the Marie-Curie Training Network
CanTrain (CT-MC-RTN-2004-512481), a OeAW-DOCfFORTE PhD Studentship to OM, and
a VBC PhD Programme fellowship to IF.




                                             169
P120C
Neutrophil extracellular trap formation releases S100 proteins crucial for antifungal
immune responses
Constantin F. Urban1, David Ermert2, Monika Schmid2, Ulrike Abu-Abed2, Wolfgang
Nacken3, Volker Brinkmann2, Peter R. Jungblut2 and Arturo Zychlinsky2
1 Molecular Biology, Umeå University, Sjukhusområdet 6 KL, Umeå 90187, Sweden, Phone:
+46 (0)90 785 3341, FAX: +46 (0)90 772630, e-mail: constantin.urban@molbiol.umu.se,
Web: www.molbiol.umu.se
2 Max Planck Institute for Infection Biology, Berlin, Germany
3 Experimental Dermatology, Muenster University, Muenster, 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 antimicrobial proteins and chromatin that together form
extracellular fibres, called Neutrophil Extracellular Traps (NETs). We showed that these NETs
can ensnare and kill C. albicans yeast and hyphal forms. Moreover, we found that hyphae are
more potent to induce NET formation. This is interesting because hyphae are the large and
invasive forms that cannot be engulfed by a single neutrophil.
To understand how NETs kill C. albicans on the molecular level we analyzed all proteins
bound to NETs. We isolated the released NETs and identified 24 NET-associated proteins by
mass spectrometry. We identified a protein from the S100 family as the major antifungal
protein in NETs. We demonstrated that NETs with associated S100 protein are present in vivo
during C. albicans infection. To investigate the contribution of the protein to the antifungal
response in vivo we compared wild type and knockout mice in different C. albicans infection
models. The knockout animals were significantly more susceptible towards C. albicans
challenge. We conclude that NETs and NET-associated S100 proteins are essential for
efficient antifungal immune responses.




                                           170
P121A
A patho-assay using S. cerevisiae and C. elegans reveals novel roles for yeast AP-1, Yap1
and host dual oxidase BLI-3 in fungal pathogenesis
Charu Jain, Meijiang Yun, Samuel Politz and Reeta Prusty Rao
Biology & Biotechnology, WPI, 100 Institute Road, Worcester MA 01609, USA, Phone: 001-
508-831-6120,        FAX:       001-508-831-5936,    e-mail:      rpr@wpi.edu,       Web:
http://users.wpi.edu/~prustyraolab/

Treatment of systemic fungal infections is difficult because of the limited number of
antimycotic drugs available. Thus, there is an immediate need for simple and innovative
systems to assay the contribution of individual genes to fungal pathogenesis. We have
developed a patho-assay using Caenorhabditis elegans, an established model host and
Saccharomyces cerevisiae as the invading fungus. We have found that yeast infects nematodes
causing disease and death. Our data indicates that the host produces reactive oxygen species
(ROS) in response to fungal infection. Yeast mutants sod1 and yap1, which cannot withstand
ROS, fail to cause disease, except in bli-3 mutant worms that have a defective oxidase.
Chemical inhibition of the NADPH oxidase domain abolishes ROS production in worms
exposed to yeast. This patho-assay is useful for conducting systematic, whole-genome screens
to identify fungal virulence factors as alternative targets for drug development and exploration
of host responses to fungal infections.




                                            171
P122B
Cbp1 is required for lysis of host macrophages by the fungal pathogen Histoplasma
capsulatum.
Dervla Isaac*, Charlotte Berkes* and Anita Sil
Microbiology and Immunology, University of California - San Francisco, 513 Parnassus Ave,
Box 0414, San Francisco CA 94143, USA, Phone: 1 415 502 4810, FAX: 1 415 476 8201, e-
mail: Dervla.Isaac@ucsf.edu

Histoplasma capsulatum is a fungal respiratory pathogen that infects mammalian host
macrophages. Successful infection is characterized by intracellular replication of the fungus
followed by macrophage lysis. To date, however, very little is known about how Hc interacts
with the macrophage to trigger host cell death. The secreted factor Cbp1 has previously been
shown to be required for host colonization and virulence. Using a cbp1 insertion mutant, we
show that CBP1 is dispensible for Hc growth in bone marrow derived macrophages
(BMDMs), but not for BMDM lysis. The cbp1 mutant replicated to higher levels within
macrophages than wild-type Hc, but the mutant was incapable of triggering macrophage lysis.
Complementation with the CBP1 gene restored the ability of the mutant cells to lyse host
macrophages. To further understand the role of Cbp1 during infection, we examined the
transcriptional profile of macrophages infected with wildtype Hc or the cbp1 mutant. We
identified a host transcriptional signature that was induced specifically by live wildtype Hc in
a Cbp1-dependent manner. These data suggest that Cbp1 promotes a specific transcriptional
program that correlates with lysis of host macrophages.

•   = authors contributed equally to this work




                                            172
P123C
Fungal gigantism during mammalian infection
Oscar Zaragoza1, Josh Nosanchuk2, Manuel Cuenca-Estrella1, Juan Luis Rodriguez-Tudela1
and Arturo Casadevall2
1 Mycology Department, National Center for Microbiology, ISCIII, Crta. Majadahonda-
Pozuelo, Km2, Majadahonda, Madrid 28220, Spain, Phone: + 34 91 822 3661, FAX: + 34 91
509 70 34, e-mail: ozaragoza@isciii.es
2 Albert Einstein College of Medicine. 1300 Morris Park Avenue. Bronx, New York 10461

Morphological changes are common features among fungal pathogens during the interaction
with the host. Cryptococcus neoformans has become one of the main model microorganisms to
study fungal infections and host-pathogen interactions in the last years. One of its main
characteristics is the presence of a polysaccharide capsule that surrounds the cell body, which
is a unique feature among fungal pathogens. Although filamentous growth does not seem to
play a role in cryptococcal pathogenesis, this fungus undergoes two morphological changes
during the interaction with the host which occur at different moments. During the first hours of
infection, the capsule suffers a significant increase in its size, which is considered an early
morphological response. The other morphological change is observed after three-four weeks of
infection (late morphological change), and involves the formation of “giant” cells. The
diameter of these cells can reach up to 70 microns, and its elimination poses a problem for the
immune system. In the laboratory we are interested in these morphological changes, with
special attention to giant cell formation. The volume of giant cells is around 900-fold larger
compared to cells grown in vitro, which suggests that giant cell formation is a high-energy cost
process. The capsule, which is the main virulence factor of this organism, suffers a significant
increase in the density and packing of the polysaccharide fibbers. During infection, we found
that the proportion of giant cells could be very variable, ranging from 5 to 90% of the total
fungal population. Although we have not identified the factors that influence the proportion of
giant cells in vivo, our data suggests that host factors play a key role in this process.
Interesting, we found an inverse correlation between the proportion of giant cells and
inflammation degree in the lung. Our data suggest that gigantism plays an important role
during the interaction of Cryptococcus neoformans with the host, and that it is a strategy
developed by this fungus that allows immune response evasion and prolonged survival in the
host.




                                            173
P124A
Comprehensive gene deletion study to identify cell wall organisation and structure in
Candida glabrata
Rebecca Stevens1, Ekkehard Hiller1, Marcel Dörflinger1, Toni Gabaldon2, Tobias
Schwarzmüller3, Karl Kuchler3 and Steffen Rupp1
1 MBT, Fraunhofer IGB, Nobelstrasse 12, Stuttgart 70569, Germany, Phone: +49 (0)711-970-
4048, FAX: +49 (0)711-970-4200, e-mail: Rebecca.Stevens@igb.fraunhofer.de, Web:
www.igb.fraunhofer.de
2 CRG, C/ Dr. Aiguader, 88, 08003 Barcelona, Spain
3 Medical University Vienna, Max F. Perutz Laboratories, Department of Medical
Biochemistry, Dr. Bohr-Gasse 9/2, 1030 Vienna, Austria

Although Candida glabrata has become the second most important pathogenic Candida
species, only few of its virulence mechanisms have been identified so far.
To get a more comprehensive idea of the virulence mechanisms of C. glabrata, we use
comprehensive gene deletion studies in order to elucidate the organisation and components of
its cell wall. These studies are undertaken within an ERA-Net consortium, FunPath. Genes
coding for putative proteins of the cell wall, known signalling pathways, membrane-bound
receptors, transporters and transcription factors were identified by comparative genome
analysis and subsequently deleted (about 500 deletion mutants at present). This library is
screened with biological assays for strains with altered cell wall stability, stress tolerance, or
adhesion.
Up to now, several strains were found in survival assays on plates to be more sensitive to
congo red, osmotic stress or increased temperature. The genes deleted in these mutants were
homologues to Saccharomyces cerevisiae genes that are involved in cell wall integrity and the
MapK-pathways.
The ability to adhere on a surface is tested by a series of tests with increasing complexity and
approximation to the host environment. To get a first hint of the adherence ability of the
mutants we analysed their adhesion on solid agar plates using wash tests. The genes we
identified to be important for adherence under these conditions were associated to the cell wall
or involved in cytokinesis. To verify our screening results we will investigate the adhesion and
invasion behaviour of the interesting mutant strains in in vitro experiments with a human
epithelial model. In addition to the screening we plan to analyse the adhesion ability of all
mutant strains in a comparative approach with pools of mutants on epithelial models. All
deletion strains are tagged with a specific barcode sequence that can be detected via an in-
house barcode microarray. Experiments with in vivo models will be carried out by our project
partners (FunPath). Using genome wide transcription profiles, it will be possible, for instance,
to further characterize strains with reduced virulence. The results generated will allow
conclusions about basic pathogenicity mechanisms and possible targets for the therapy of
fungal infections.




                                             174
P125B
A tool for analysis of N-glycosylation in Candida albicans
Shahida Shahana, Hector Mora-montes, Castillo Luis, Chirag C. Sheth, Frank C. Odds, Neil
A. Gow and Alistair J. P. Brown
Aberdeen fungal group, Institution of Medical Sciences, Foresterhill, Aberdeen Ab25 2ZD,
UK, Phone: +44(0)1224 555878, FAX: +44(0)1224 555844, e-mail: s.shahana@abdn.ac.uk

Background: Recognition of Candida albicans by host cells is based on its cell wall
component, which is considered to be responsible for its virulence nature. Cell wall glycans
play an important role in regulation of balance between saprophytism and parasitism, and also
between resistance and infection. Candida albicans is able to regulate its glycan surface
expression. At the same amino acids within a population of protein molecules may express an
array of different carbohydrate structures. Due to this site-specific heterogeneity
characterization of glycosylation is complicated. Method: To minimise this problem we
developed a molecular tool to generate homogenous population of N-glycans in Candida
albicans. The reporter contains a single N-linked glycosylation site and is tagged with FLAG
and His6 at its C terminal domain for identification and purification, respectively.
Saccharomyces cerevisiae Suc2 and Candida Albicans Sap2 sequence were re-engineered to
design the construct. Sap2 N-terminal region was used to allow the secretion of the protein.
Results: The reporter protein is expressed and secreted by wild type strain of Candida
Albicans and N-glycosylation was confirmed by Endoglycosidase H (which cleaves
asparagine-linked mannose rich oligosaccharides) treatment. Conclusion: This tool can be
useful for the characterization of N-glycan and also to study N-glycosylation pathway in any
Candida species.

Acknowledgement: We are thankful to welcome trust for funding our work.

Reference:
1. Martínez-Esparza M et al., (2006), J Immunol Methods, 314(1-2), page90
2. Medzihradszky KF el al., (2008), Methods Mol Biol.,446, page293
3. Li H et al., (2007), ,389, page139




                                          175
P126C
Candida glabrata infection: alternative host models and the role of calcium signalling
Lucy Holcombe1, Eimear Jackson1, Marlies Mooij2, Fergal O'Gara2 and John Morrissey1
1 Department of Microbiology, University College Cork, College Road, Cork City Co. Cork,
Ireland, Phone: +353 (0)21 4902934, FAX: +353 (0)21 4903101, e-mail: l.holcombe@ucc.ie,
Web: http://www.ucc.ie/en/tramways/research/Fungal/
2 Biomerit Research Centre, University College Cork, Cork, Ireland

Candida glabrata infections are linked to high levels of mortality and morbidity among
immunocompromised individuals. Many of the fungal pathogenicity factors which are required
for virulence in mammals have also been shown to be important for fungal survival during
infection of invertebrate hosts, making these relatively simple organisms appropriate for use in
virulence studies. We have compared macrophage, Acanthamoeba, Galleria and zebrafish
hosts to evaluate their potential as alternative model organisms for the study of C. glabrata
infection. Each model exhibits different characteristics of host immunity providing a spectrum
of early infection responses ranging from those involved in host phagocytosis to those
stemming from innate immunity. As virulence is often influenced by the perception and
transduction of signals through key stress response pathways, we extended our analysis to
investigate the role of the calcium signalling pathway in C. glabrata pathogenicity. Calcium-
dependent signalling mechanisms are thought to be involved in the regulation of a wide variety
of fungal responses to stress including survival in the host environment and resistance to anti-
fungal drugs. In the presence of an external stress calcium enters the cell via plasma membrane
channels encoded by genes including MID1, CCH1 and FIG1 and activates the calcium-
binding protein calmodulin, this in turn activates calcineurin, a protein phosphatase
responsible for the stimulation of downstream target genes including the transcription factor
Crz1. This stress response pathway, which has been extensively studied in the model yeast
Saccharomyces cerevisiae, appears to be widely conserved amongst pathogenic fungi. Using
deletion mutants we have analysed the roles of some of the calcium signalling components in
C. glabrata stress response and survival.




                                            176
P127A
Profiling of host responses in Candida parapsilosis infection
Zsuzsanna Hamari1, Joshua D. Nosanchuk2, Csaba Vagvolgyi1 and Attila Gacser1
1 Department of Microbiology, University of Szeged, Kozep fasor 52, Szeged 6726, Hungary,
Phone: +36 62 544849, FAX: +36 62 544823, e-mail: gacsera@gmail.com
2 Albert Einstein Collage of Medicine, Department of Microbiology and Immunology, 1300
Morris park ave., Bronx, NY 10461 USA

Over the past decade, Candida parapsilosis has become a major human pathogen. In fact, C.
parapsilosis is now the second most commonly isolated Candida species from blood cultures
worldwide, and C. parapsilosis even outranks C. albicans in some European hospitals. Despite
the alarming increase in the incidence of invasive C. parapsilosis disease, little is known
regarding the molecular and structural basis of virulence or the regulation of genes involved in
the host response to the pathogen.
Lipases hydrolyse ester bonds at the interface between insoluble substrates. Putative roles of
microbial extracellular lipases in microbial virulence include the digestion of lipids for nutrient
acquisition, adhesion to host cells and tissues, synergistic interactions with other enzymes,
nonspecific hydrolysis, initiation of inflammatory processes by affecting immune cells, and
self-defense by lysing the competing microflora. We previously showed that C. parapsilosis
LIP1-LIP2 knockout mutants were significantly deficient in their capacity to produce biofilm,
to grow in lipid rich medium, and to survive in macrophages. In an attempt to understand this
reduced virulence phenotype, we examined the gene expression in BALB/c mice infected with
wild type C. parapsilosis and lipase minus cells. Invasive fungal infection was modeled by
intravenous infection of BALB/c mice with fungal cells. Invasive infection was allowed to
progress for 30 minutes and 1 hour, and total mouse RNA was then extracted from white
blood cells. The labeled cDNA was hybridized to Agilent Mouse Whole Genome 44k Arrays.
The data analysis is in progress and we are focusing on differential transcriptional regulation
and affects on signaling pathways. Selected genes encoding interleukins, chemokine ligands,
receptors, other interesting signal transduction pathway members and cytokines will be
confirmed by quantitative real-time reverse transcriptase polymerase chain reaction.
Exploration of host responses to C. parapsilosis infection might yield novel insights into the
pathogenesis of disease that may identify targets for the development of therapeutics to
efficiently combat this emerging fungal pathogen.




                                             177
P128B
Autophagy is a macrophage immune mechanism against intracellular parasitism by
Cryptococcus neoformans
André Moraes Nicola, Rafael Antonio Dal-Rosso, Patrícia Albuquerque de Andrade and
Arturo Casadevall
Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park
avenue, Bronx NY 10461, USA, Phone: +1-718-430-3766, FAX: +1-718-430-8701, e-mail:
anicola@aecom.yu.edu

Cryptococcus neoformans is an encapsulated yeast that is a frequent cause of meningitis in
immunocompromised people. Macrophages are major effector cells in the immune response to
this infection, as is the case with other facultative intracellular pathogens. Unlike other
pathogens, C. neoformans does not inhibit phagolysosomal maturation nor escapes from the
cryptococcal vacuole (CnV), but rather survives and even replicates in the harsh environment
of the phagolysosome. This fact implies that macrophages must have an additional mechanism
to control phagocytosed C. neoformans. We have hypothesized that autophagy might be this
mechanism. Autophagy is a conserved tool used by eukaryotes to recycle cellular material. It
has been recently involved in control of intracellular pathogens, such as viruses, bacteria and
protozoa. To test the hypothesis, we first used immunofluorescence to determine that LC3, a
marker of autophagy, is present in the CnV both in J774 macrophage-like cells and primary
murine peritoneal macrophages. Time-lapse confocal imaging using J774 cells transfected
with LC3 coupled to enhanced green fluorescent protein (LC3-EGFP) was used to study the
formation of such vacuole. Instead of the traditional cup-shaped sequestration membranes,
hallmarks of autophagosome formation, this giant autophagosome appeared to form by
sequential fusion of small autophagic vacuoles (Av) with the CnV. These fusion events were
also apparent by transmission electron microscopy, which revealed fusion of the outer
membrane of double-membraned vesicles with the CnV. To determine what role autophagy
plays in the control of intracellular C. neoformans, we generated autophagy-deficient J774
cells by stable transfection with shRNAs targeting ATG5, a gene that is essential for
autophagy. These cells were infected with C. neoformans and plated for colony forming units
after 24h, to count viable cells. This experiment revealed that ATG5 knockdown decreased the
ability of the J774 cells to restrict intracellular growth of the fungus in a dose-dependent
manner. In conclusion, we have observed that the CnV acquires the autophagy marker LC3
after maturation and that this autophagosome is not formed by the typical sequestration
membranes. Furthermore, functional studies demonstrate that this autophagosome is necessary
for efficient restriction of infection by C. neoformans.




                                           178
P129C
Characterization of genes encoding for cell surface proteins induced during 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. Its
presence becomes a problem in immunocompromised patients where it may turn into an
opportunistic pathogen. Candida colonizes various host niches including skin, gastrointestinal
and the urogenital tract, which offer various environments in terms of pH and nutrient
availability. The cell surface of the fungus is the site of direct interaction of Candida with the
host, mediating the environmental sensing, adhesion and also interaction with the host immune
system. Since the cell wall is not present in humans its components are a prime target for drug
development. To reveal whether C. albicans is able to specifically react to different epithelial
tissue or other surfaces in a specific manner we used transcriptional profiling in order to
identify genes differentially regulated during adhesion focusing on cell surface proteins. One
of the genes identified was termed Adhesion Upregulated Factor – AUF8. The respective
protein is predicted to be localized in the plasma membrane carrying four transmembrane
domains. AUF8 is upregulated in an adhesion dependent manner with the strongest induction
on intestinal tissue model after two hours of interaction. For AUF8 we identified six
homologues in the C. albicans genome, of which five together with AUF8 are in one 10 kb
gene-cluster. When heterologously expressed in S. cerevisiae Auf8p is localized to the plasma
membrane. Deletion studies indicate that the AUF genes may be required for fitness during
stationary phase. However, its function during adhesion is still unclear. Other proteins encoded
by genes upregulated during adhesion and first steps of invasion are localized in the cell wall.
The functional studies of PGA7, PGA23 and PRA1 with regards to adhesion and invasion as
well as cell wall stability were performed using deletion and overexpression strains. Our
studies qualify PGA7 and PGA23 as structural elements of the cell wall rather than adhesins.




                                             179
P130A
Candida albicans secreted aspartyl protease expression influence T cell function in a
model of murine peritonitis
Alexandra Correia1, Íris Caramalho2, Patrícia Meireles3, Ulrich Lermann4, Paula Sampaio1,
Joachim Morschhaueser4, Célia Pais1 and Manuel Vilanova5
1 Centro de Biologia Molecular e Ambiental, Universidade do Minho, Campus de Gualtar,
Braga 4710 057, Portugal, Phone: +351 253604310, FAX: +351 253678980, e-mail:
alexandracorreia@bio.uminho.pt, Web: http://www.cbma.bio.uminho.pt
2 Instituto Gulbenkian de Ciência, Oeiras, PORTUGAL
3 Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, PORTUGAL
4 Institut fur Molekulare Infektionsbiologie, Universitat Wurzburg, Wurzburg, GERMANY
5 Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, PORTUGAL;
Instituto de Biologia Molecular e Celular, Porto, PORTUGAL

Host immune response to Candida albicans acquired a renewed interest due to the dramatic
increase in the incidence of candidiasis in immunocompromised hosts. Several virulence
factors are thought to participate in the infective process, including the secreted aspartyl
proteases (Sap). Studies using gene deficient mutant strains highlighted the importance of
specific SAP genes on the virulence of C. albicans in different models of infection. However,
the effect of Sap deficiency on the elicited immune response to acute systemic candidiasis and
peritonitis is mostly unknown. In this study, we investigated whether lack of Sap1-3 and Sap4-
6 expression could affect both innate and acquired immunity of BALB/c mice, 3 and 7 days
after intravenous or intraperitoneal infection with sub-lethal inocula of C. albicans wild-type
(WT) strain SC5314 or its derived SAT1-flipping triple mutants sap123 and sap456.
Analysis of intracellular expression of Foxp3 in splenic CD4+CD25+ T cells, 3 days after
peritoneal challenge, revealed a significant down-regulation of this marker in mutsap456-
infected mice and consequently higher Teffector/Treg ratios, comparatively to naïve and other
infected mice. The effector function of these cells was evaluated by using a CFSE-based
suppression assay. Sorted splenic CFSE-stained naïve (CD4+CD25-) T cells, co-cultured with
CD4+CD25+ T cells from naïve and infected mice, were stimulated with anti-CD3 and
analysed by FACS. Lack of SAP4-6 genes resulted in impaired Treg cell mediated suppression
of CD4+CD25- T cells proliferation. Additionally, IFN-&#947;, IL-4 and IL-10 cytokine
levels were assessed in the supernatants of anti-CD3 stimulated sorted splenic CD4+ T and
CD4+CD25- T cells from naïve and infected mice. Both CD4+ T cell subsets from WT-
infected mice produced the highest levels of IL-10, which is known to be implicated in the
control of Th1-mediated immunity to the fungus. In contrast, no significant differences could
be found in the immune response elicited in the spleens of i.v.-infected mice by any of the
strains analysed.
Altogether, these results implicate Sap expression, in particular Sap4-6, in the modulation of
the host immune response to C. albicans peritonitis, but not to acute systemic candidiasis. This
study provides additional evidence for the differential role of these secreted proteins as
virulence determinants in different infection models.

This work was supported by POCTI/SAU-IMI/58014/2004 and SFRH/BD/31354/2006.




                                            180
P131B
Comparative study of immune response to HSP70 protein of fungal and mouse origins in
Aspergillus fumigatus induced asthma mouse model
Elena Shekhovtsova, Marina Shevchenko and Alexander Sapozhnikov
Immunology, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-
Maklaya st., 16/10, Moscow 117997, Russia, Phone: +7 495 330 40 11, FAX: +7 495 330 40
11, e-mail: shehovcova_elena@mail.ru

Ubiquitous fungi Aspergillus fumigatus (Af) are able to cause severe allergic disease,
characterized with the increasing of specific IgG1 and IgE production. In some cases
antifungal antibodies were reported to be also cross-reactive to self proteins showing high
level of homology to Af antigens. HSP70 is a highly conservative family of chaperone
proteins. Cross reactivity to self HSP of serum antibody was also shown in patients, suffering
with atopic exema/dermatitis syndrome and sensitized with Mala s 10 – member of HSP70
family.
The aim of this study was to detect the difference between humoral and cell response to
exogenously injected HSP70 protein from Af and mouse HSP70 during asthma establishment
in a mice model.
HSP70 was derived from Af culture filtrate (AfHSP70) or from liver and kidney homogenate
of syngeneic mice (mHSP70) and purified on ATP-agarose column. BALB/c mice received
correspondent antigen i.p. in PBS without any adjuvant, in dose 10ug/mouse/injection
multiply. A week after last injection mice were received Af crude extract (Af crude)
125ug/mouse/time, 3time/day, 3days/week i.n. Serum specific IgG1 production and cross-
reactivity of antibody, specific to HSP70 of different origin were detected by ELISA assay.
Lung inflammation was estimated by BAL cell count.
Af HSP70 demonstrated increasing of IgG1 production on a 3rd week after beginning of
immunization. Immunization of mice with mHSP70 didn’t induce any specific antibody
production in the same time frames. There was no detected cross-reactivity of AfHSP70
specific antibody to mHSP70. Af crude inhalation didn’t induce any lung inflammation in mice
which were receiving 10 injections of mHSP70, whereas mice, immunized with Af HSP shown
slight lung inflammation, characterized by 0,3+/-0,05 mln macrophages infiltration. The level
of specific IgG1 in mice immunized with mHSP70 was increasing only as a result of the long
time immunization (daily, within 2 month). Lung inflammation induced by Af was
significantly enhanced in mice, underwent long term immunization with mHSP70 (17+/-0,7
mln cells) compare to mice, sensitized for a long time with AfHSP70 or OVA (1+/-0,3 mln
cells). BAL infiltrates in mice, immunized with self HSP70 were dominantly represented by
neutrophils, which number was more then 10 fold higher compared with mice, immunized
with OVA or Af HSP70.
So HSP70 from A. fumigatus is able to initiate immune response but unable to skew B cells to
autologous antibody production.




                                           181
P132C
A. fumigatus conidia inhalation significantly alters severity and character of lung
inflammation in a mouse model of allergic aspergillosis
Marina Shevchenko, Elena Shekhovtsova and Alexander Sapozhnikov
Immunology, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya
16/10, Moscow 117997, Russia, Phone: +74953304011, FAX: +74953304011, e-mail:
shev@mx.ibch.ru, Web: http://www.ibch.ru/

A. fumigatus conidia (Af conidia) are able to induce lung inflammation results in exogenous
alveolitis in a healthy man and in severe asthma or allergic bronchopulmonary aspergillosis.
In this study we aimed to investigate the influence of Af conidia on allergic asthma
inflammation in a mouse model.
Mice were sensitized i.p. with chicken ovalbumin (OVA) multiply in phosphate buffer without
any adjuvant in a doze 10ug/mice/injection. Challenge with Af crude performed i.n. in a doze
100ug/mice/time 3 times a week. Af conidia were implied for mice i.n. in a single dose 1*107
conidia/mouse. For invasive aspergillosis model mice received cyclophosphamide and
cortisone acetate according standard procedure and i.n. challenged with Af conidia in dose
1*107 conidia/mouse. Specific antibody level detected by ELISA. Lung infiltrating detected
by total and differential cell count in bronchoalveolar lavage (BAL).
Single challenge with conidia doesn’t influence antibody production in nonsensitized or
immunosuppresed mice, but enhance IgE production in sensitized mice. When in BALs of
challenged with Af crude sensitized mice dominant cells are macrophages, Af conidia
challenge induce significant influx of neutrophils. The number of neutrophils in this case in 10
folds increase compare to Af crude challenged sensitized mice and 5 folds higher then that in
Af conidia challenged non sensitized animals. Interestingly, in mice, undergoing
immunosuppression according to invasive aspergillosis model protocol, in the absence of
neutrophils the number of macrophages was three folds elevated compare to
immunocompetent sensitized or non sensitized animals.
Thus, inhalation of A. fumigatus conidia induces significant neutrophils influx to the lung, but
also enhances proallergic immunoglobulin production in case of prior sensitization.




                                            182
P133A
A role for Aspergillus terreus accessory conidia in virulence during infection
Eszter Deak1, Chad Steele2, John Baddley3 and S. Arunmozhi Balajee1
1 Mycotic Diseases Branch, Centers for Disease Control, 1600 Clifton Rd., Atlanta GA 30333,
USA, Phone: 404-639-1342, FAX: 404-639-3546, e-mail: guf0@cdc.gov
2 Department of Medicine and Microbiology, University of Alabama at Birmingham School
of Medicine
3 Division of Infectious Diseases, University of Alabama at Birmingham School of Medicine

Infection with Aspergillus terreus results in more invasive, disseminated disease when
compared to other Aspergillus species; importantly this species appears to be less susceptible
to the antifungal drug amphotericin B (AMB). Unique to this species is the ability to produce
specialized structures denoted as accessory conidia (AC) directly on hyphae both in vitro and
in vivo. With the hypothesis that production of AC by A. terreus may enhance virulence of this
organism, we analyzed the phenotype, structure and metabolic potential of these conidia.
Comparison of A. terreus phialidic conidia (conidia that arise from conidiophores, PC) and AC
architecture by electron microscopy revealed distinct morphological differences between the
two conidial forms; AC have a smoother, thicker outer cell surface with no apparent pigment-
like layer. Further, AC germinated rapidly, had enhanced adherence to microspheres, and were
metabolically more active compared to PC. Additionally, AC contained less cell membrane
ergosterol, which correlated with decreased susceptibility to AMB. Furthermore, AC were
enriched at their tips with beta 1-3 glucan, suggestive of attachment scarring. AC were
resistant to phagocytosis by human macrophages and elicited a inflammatory response when
exposed to macrophages. Collectively, this study suggests that A. terreus accessory conidia
exhibit candidate virulence factors – adherence, excellent viability, rapid germination potential
and ability to deter monocyte phagocytosis.




                                            183
P134B
Discrimination of Yeast from Hyphal States of Candida albicans by Oral Epithelial Cells
involves MAPK signalling, MKP-1 and c-Fos
David Moyes, Manohursingh Runglall, Celia Murciano, Stephen Challacombe and Julian
Naglik
Oral Immunology, King's College London, St Thomas Street, London SE1 9RT, UK, Phone:
+44 20 7188 4377, FAX: +44 20 7188 4375, e-mail: julian.naglik@kcl.ac.uk

The mucosal epithelium has immense importance in host defence and surveillance, as it is the
cell layer that initially encounters most microorganisms. Host mechanisms enabling
discrimination between commensal and pathogenic organisms are critical in mucosal immune
defense and homeostasis. The polymorphic human fungal pathogen Candida albicans can act
as a commensal or pathogen and is the most common fungal pathogen of humans. Previously,
it has been demonstrated that C. albicans hyphae rather than yeast are associated with
virulence. Here we demonstrate that oral epithelial cells orchestrate the innate response to this
adaptable fungus via NF-kB and a biphasic MAPK response dependent on recognition of
hyphae. The response of the oral epithelial cell line, TR146, to C. albicans was assessed at
different time points using a variety of parameters. C. albicans activated both the NF-kB and
MAPK pathway. Both IkBa phosphorylation and p65 DNA binding activity (NF-kB pathway)
increased with linear kinetics, whilst all three MAPK pathways (ERK1/2, JNK and p38) were
activated in a biphasic pattern with an early weak phase and a late strong phase. The early
phase peaked at 15 – 30 min post-infection and was associated with a temporary increase in c-
Jun DNA binding activity, whilst the late phase peaked at 2 h post-infection and was
associated with an increase in c-Fos DNA binding activity, stabilisation of the MAPK-
regulating phosphatase MKP-1, and a decrease in MEF2 DNA binding activity. Infection of
oral epithelial cells with non-filamentous or hyperfilamentous mutants or with pre-induced C.
albicans hyphae indicated that whilst NF-kB activation was independent of morphology,
MKP-1 and c-Fos activation were both dependent on the presence of hyphae. Comparison of
strains used in a murine model of colonisation indicated that those strains that successfully
colonise do not produce hyphae, implying that successful colonisation is dependent on the lack
of hyphal formation. Further work demonstrated that strains that successfully colonised did not
induce cytokines, cause damage or activate c-Fos and MKP-1, although they did activate NF-
kB signaling. We therefore propose a mechanism enabling epithelial cells to distinguish
between commensal (yeast) and pathogenic (hyphal) forms of C. albicans through selective
activation of MAPK signaling, MKP-1, and the c-Fos transcription factors.




                                            184
P135C
Candida albicans HGT1 is a major complement FH-, and C4BP-binding molecule
Iwona Lesiak1, Georgia Vogl2, Tobias Schwarzmüller1, Cornelia Speth2, Cornelia Lass-Flörl2,
Manfred P. Dierich2, Karl Kuchler1 and Reinhard Würzner2
1 Molecular Genetic, Medical Biochemistry, Dr. Bohr-Gasse 9, Vienna 1030, AUSTRIA,
Phone: +43 (0) 4277 61812, FAX: +43 (0) 4277 9618, e-mail: iwona.lesiak@univie.ac.at,
Web: www.meduniwien.ac.at/medbch/MolGen/kuchler//
2 Department for Hygiene, Microbiology and Social Medicine, Innsbruck Medical University,
Fritz-Pregl-Strasse 3, 6020 Innsbruck, AUSTRIA

Candida albicans is a ubiquitous saprophyte of mucous membranes, predominantly colonising
the gastrointestinal tract. Complement is an important part of the innate immunity against
infection by facilitating, among other effects, chemotaxis and opsonisation, sometimes
followed by lysis of the intruder. This cascade system is tightly controlled by several fluid
phase and cellular regulators. Factor H (FH), a soluble plasma protein, is the main fluid phase
regulator of the complement alternative pathway, whereas C4b-binding protein (C4BP) is the
classical pathway regulator. Both proteins can be acquired onto the surface by a number of
human pathogens conveying resistance to complement and thus contributing to their
pathogenic potential. „High affinity glucose transporter 1“ of C. albicans Cahgt1 was
identified as the factor H binding molecules by an expression library. Candida albicans
hgt1/hgt1 mutants constructed using the fusion PCR protocol confirmed its role as major factor
H-binding protein as these mutants showed a markedly decreased binding of factor H and also
of C4BP. Reduced binding of complement regulators to C. albicans, hgt1/hgt1 mutants led to
an increased complement activity (terminal complement complex deposition) after incubation
with human serum at 37°C when compared to wild type and parental strains. The ability of the
mutants to form hyphae at 37°C was decreased in comparison to the wild type. hgt1/hgt1
mutants were also not able to form rosettes with complement-coated sheep erythrocytes when
compared to wild type or parental strain, implying that C3R-like moiety is lacking. This study
confirmed the role of Cahgt1 not only as FH-, but also as C4BP- binding molecule, and also as
CR3 analogue and revealed the importance of the HGT1 gene in modulation of other virulence
factors in Candida albicans, such as hyphae formation.




                                           185
P136A
Analysis of Candida albicans cell wall glycans during phagocytosis by macrophage
Aurore Sarazin1, Maria Martínez-Esparza2, Daniel Poulain1 and Thierry Jouault1
1 Inserm U799-Mycologie Fondamentale et Appliquée, Faculté de médecine, pole recherche,
Place Verdun,Avenue Oscar Lambret, Lille 59045, FRANCE, Phone: +33 (0) 3 20 62 34 15,
FAX: + 33 (0) 3 20 62 34 16, e-mail: aurore.sarazin@laposte.net
2 Departamento de Bioquímica B e Inmunología, Facultad de Medicina. Universidad de
Murcia, Murcia, Spain

Recognition of yeasts by macrophages is based on components of the yeast cell wall, which
are considered part of its virulence attributes. Depending of the availability of the different cell
wall glycans, either alpha- or beta-mannosides or beta-glucans, immune cells response is
directed differently. In this work we exploited flow cytometry methods to probe and the
availability of surface glycans before and during phagocytosis by macrophages.
Yeast blastoconidiae, either C. albicans or S. cerevisiae, or heat-killed yeasts were incubated
at different yeasts : cell ratio for different time with macrophages. Exposition of yeast surface
glycans was then evaluated after recovery of ingested yeasts by monoclonal or polyclonal
antibodies specific for beta-mannosides, alpha-mannosides, and beta-glucans or lectin for
chitine.
Expression levels of alpha- and beta-linked mannosides as well as beta-glucans were
successfully evaluated by flow cytometry. Glycans were shown to be differently presented at
the yeast surface during the course of phagocytosis by macrophages. Exposure of the different
glycans was dependent on the strains examined either C. albicans or S. cerevisiae, on heat-kill
treatment and on the yeast : macrophage ratio (1:1 or 10:1). For C. albicans, levels of beta-
mannosides as well as alpha-mannosides present at the yeast surface when in proportion 10:1,
increased during phagocytosis by macrophages, whereas beta-glucans and chitine were not
accessible. In contrast, when a 1:1 ratio was used, levels of both beta-mannosides and alpha-
mannosides decreased during phagocytosis. In parallel, beta-glucans and chitine were detected
after 1h of incubation, with a maximum after 4h. For S. cerevisiae, presence of both types of
mannoside decreased with an increased amount of accessible beta-glucans at the yeast surface
after 1h of phagocytosis. In contrast to live yeast cells, when using heat killed C. albicans or S.
cerevisiae blastoconidiae, no difference was observed between 1:1 and 10:1 ratios.
Together these results show that depending on the C. albicans burden, glycans are differently
accessible to innate immune receptors during phagocytosis by macrophages. However when
heat killed yeasts which are not able to modulate their cell wall components, were used, no
difference could be observed suggesting that glycans availability during phagocytosis may
result from a response of yeasts to the phagosome environment.




                                              186
P137B
Treatment of Pneumocystis pneumonia with echinocandins selectively depletes cysts with
large populations of trophic forms remaining: implications for life cycle
Melanie T. Cushion1, Michael J. Linke2, Alan Ashbaugh1 and Margaret S. Collins1
1 Internal Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way,
Cincinnati OH 45267-0560, United States, Phone: +01 (513) 861 3100 ext. 4417, FAX: +01
(513)        475          6415,          e-mail:      melanie.cushion@uc.edu,       Web:
http://medportal.uc.edu/portal/default.cfm?pageset_id=117759
2 Veterans Affairs Medical Center, Cincinnati, OH

The life cycle of Pneumocystis spp. appears to involve an asexual phase in which the trophic
form replicates by binary fission and a sexual phase resulting in formation of cysts (asci).
Transmission is thought to occur via an airborne route, but the infectious agent has not been
identified. We found that treatment of Pneumocystis pneumonia (PCP) in immunosuppressed
rodents with anidulafungin, caspofungin and micafungin resulted in dramatic depletion of
cysts across a broad spectrum of doses. However, trophic forms persisted in high numbers
with sometimes little detriment to the host. Prophylaxis experiments using a natural mode of
infection showed that caspofungin and anidulafungin treated mice exposed to untreated,
infected mice resulted in reduced infections in the treated mice that had few to no cysts, but
significant numbers of trophs. To test the hypothesis that the cyst is the infective form of P.
murina (Pm), the ability of anidulafungin-treated mice to transmit the infection by a natural,
airborne model was evaluated by microscopy and RT-PCR. Immunosupressed Pm-naive mice
that were exposed to mice treated for 3 wk with 5mg/kg anidulafungin for 2 wk did not
develop Pm infection (no PCR signal, no Pm by microscopy) whereas Pm-naive
immunosuppressed mice exposed to untreated Pm-infected control mice contracted a robust
infection (1-, 50, and 800 Pm nuclei/rx at 2, 4, 6 wk; and 10e5 and 10e6 cysts/lung at 4, 6 wk).
Trophic forms could propagate the infection if inoculated by intratracheal intubation into Pm-
naive immunosuppressed mice, but to a lesser extent than cyst-replete populations (1-, 48- and
1000 nuclei/rx at 2, 4, 6 wk vs 20, 200 and 6000 nuclei/rx; and 6x10e4 and 10e6 cysts/lung at
4 and 6 wk vs 7x10e5, 8.5x10e6 cysts/lung for control inoculated mice). These studies provide
evidence that the cyst is required for natural, airborne transmission and that trophic
populations were viable after treatment with the echinocandins but could not transmit the
infection.




                                            187
P138C
Genome-wide mapping of the coactivator ADA2 yields insight into the functional roles of
SAGA/ADA complex in Candida albicans
Adnane Sellam, Christopher Askew, Elias Epp, Hugo Lavoie, Malcolm Whiteway and Andre
Nantel
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount,
Montreal QC H4P 2R2, Canada, Phone: +1 (514) 496-6370, FAX: +1 (514) 496-9127, e-mail:
andre.nantel@nrc-cnrc.gc.ca

The SAGA/ADA coactivator complex is widely conserved throughout eukaryotes and it
regulates numerous cellular processes by coordinating histone acetylation. Analysis of the
Candida albicans genome revealed that the components of this complex are well conserved.
Here, we unravel the multiple functions of SAGA/ADA in C. albicans by determining the
genome-wide occupancy of ADA2 using Chromatin Immunoprecipitation-CHIP. We found
that Ada2p is recruited to 199 promoters upstream of genes involved in different stress-
response categories and metabolic processes. Phenotypic and transcriptomic analysis of ada2
mutants showed that Ada2p is required for the responses to oxidative stress, as well as to
treatments with tunicamycin, menadione and fluconazole. We also reveal that Ada2p
recruitment to the promoter of oxidative resistance genes is mediated by the transcription
factor Cap1p. Coactivator functions were also established for Gal4p which recruits Ada2p to
the promoters of glycolysis and pyruvate metabolism genes. Cooccupancy of Ada2p and the
drug resistance regulator Mrr1p on the promoters of core resistance genes characterizing drug
resistance in clinical strains was demonstrated. Furthermore, ADA2 deletion causes a clear
decrease in the in vivo H3K9 acetylation level of tatget genes thus illustrating its importance
for HAT activity. Finally, we used a mouse systemic infection model to demonstrate the
importance of ADA2 in virulence.




                                           188
P139A
Virulence factor of Candida tropicalis isolated from hospitalized patients
Melyssa Negri1, Mariana Henriques1, Terezinha Svidzinski2, Joana Azeredo1 and Rosário
Oliveira1
1 Biological Engineering, University of Minho, Campus Gualtar, Braga 4710-057, Portugal,
Phone: +351 253604400, FAX: +351 253678986, e-mail: melyssanegri@deb.uminho.pt, Web:
http://www.ceb.uminho.pt/biofilm/
2 Teaching and Research in Clinical Analysis Laboratory, Division of Medical Mycology,
Universidade Estadual de Maringá, Brazil.

Candida tropicalis has been reported to be one of the Candida species which is most likely to
cause bloodstream and urinary tract infections in hospitals. Several virulence factors seem to
be responsible for C. tropicalis infections, which present high potential for dissemination and
mortality. The aim of this study was to investigate the correlation between different virulence
factors (enzymes secretion, adhesion and biofilm formation) and antifungal susceptibility of
several C. tropicalis clinical isolates. This study was conducted with 8 isolates of C. tropicalis
obtained from urine cultures (4), from blood culture (1) and from central venous catheter (1),
from patients admitted to intensive care units at the University Hospital in Maringá, Paraná,
Brazil. C. tropicalis ATCC 750 was also used, as a control. Virulence factors evaluated
included: adhesion to epithelial cells and silicone, biofilm formation and enzyme production
(hemolysins, proteinases, and phospholipases). Susceptibility to fluconazole, itraconazole,
voriconazole, and amphotericin B was also determined, by E-test. Regarding adhesion, it can
be highlighted that C. tropicalis adhered significantly more (p<0.05) to epithelial cells than to
silicone. Morevoer, it was verified that all C. tropicalis were prone to form biofilms on
silicone. Regarding C. tropicalis enzymatic activity, it was possible to verify that all isolates
were able to express total hemolytic activity on sheep-blood agar medium supplemented with
glucose. However, proteinase was only produced by two urine isolates and by the isolates from
catheter and blood and only one C. tropicalis (from catheter) was phospholipase positive. All
isolates were susceptible to voriconazole, fluconazole and amphotericin B. The largest
percentage of susceptibility-dose dependence was observed for itraconazole in 4 strains
(57.1%). Furthermore one clinical isolate (14.3%) from urine was found to be resistant to the
same compound (MIC = 1 µg/ml). Thus, it is possible to conclude that there was no direct
correlation between the virulence factors assayed (secretion of enzymes, adhesion to epithelial
cells and silicone and biofilm formation). Concerning C. tropicalis susceptibility, it was not
possible to establish any relation with Candida virulence factors as well. However, it is
important to highlight that all isolates presented one or more virulence factors.




                                             189
P140B
Construction and Analysis of Genome-Scale Protein-Protein Interaction Network of
Aspergillus fumigatus
Betul Soyler1, Alper Soyler2, Tolga Can3 and Zumrut B. Ogel1
1 Fungal Molecular Genetics and Enzymology, METU, ODTU Gida Muhendisligi Bolumu,
Ankara - 06531, TURKEY, Phone: +903122105641, FAX: +903122102767, e-mail:
betulsoyler06@gmail.com, Web: -
2 ONKOGEN Diagnostics, Ankara
3 Computer Engineering, METU

The increasing availability of genomic, proteomic and other biological data over the last years
made it possible to investigate complex structures of cellular components. It is a central
challenge of bioinformatics to use this information in discovering the functional linkages
between proteins. New methods have been devised to predict functional links between proteins
using genomic information. One of these genomic data sources is obtained by the phylogenetic
profiling technique. A phylogenetic profile describes the pattern of presence or absence of a
particular protein across a set of organisms whose genomes have been sequenced. Functional
linkages between proteins can also be detected by analyzing fusion patterns of protein
domains. A third genomic data source that correlates to functional linkages between proteins is
the gene neighbor method. These data sources can be used to infer functional linkages between
proteins with the help of computational machine learning techniques such as Bayesian
Networks or Support Vector Machines. Our goal is to construct a probabilistic high-coverage
Aspergillus species protein-protein interaction network by computational methods and
genomic information described above with the ultimate goal of integrating multiple data
sources such as microarrays, GO annotations, literature data, and high-throughput
experimental data generated in our laboratory. This study will increase understanding of key
processes playing role in genetic memory, pathogenicity and enzyme production. It will also
greatly help in understanding human metabolism development of new drugs and finding cures
for diseases caused by Aspergillus fumigatus.
The financial support is given by TUBITAK (The Scientific and Technological Research
Council of Turkey)




                                           190
P141C
Rapid acquisition of aneuploidy provides increased fitness during the evolution of
antifungal drug resistance
Anna Selmecki1, Keely Dulmage1, Carrie Ketel1, Leah Cowen2, James Anderson2 and Judith
Berman1
1 Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson H, 321
Church St. SE, Minneapolis MN 55455, USA, Phone: 612-625-1971, FAX: 612-625-5754, e-
mail: jberman@umn.edu, Web: http://www.cbs.umn.edu/labs/berman/
2 University of Toronto

The evolution of drug resistance is an important process that affects clinical outcomes. In
clinical C. albicans strains, resistance to fluconazole, the most widely used antifungal, is often
associated with acquired aneuploidy. Here we analyzed the products of an in vitro evolution
experiment, focusing on three out of six populations that acquired resistance via a specific
segmental aneuploidy, isochromosome 5L (i(5L)), in the presence of fluconazole. In all three
populations, i(5L) appeared soon after exposure to fluconazole, was associated with increased
fitness in the presence of drug, and became fixed in these independent populations. In two
cases, large supernumerary chromosomes including extra copies of Chr5L also arose during
exposure to the drug. Thus, chromosomal rearrangements that increase the dosage of genes on
Chr5L are a major source of fluconazole resistance in replicated experimental populations as
well as in their natural habitat, the human host.




                                             191
LIST OF PARTICIPANTS

        AND

 INDEX OF AUTHORS




         192
                                         List of Participants

ALBUQUERQUE DE ANDRADE Patrícia                        ANDRIANOPOULOS Alex
Microbiology and Immunology                            Department of Genetics
Albert Einstein College of Medicine                    University of Melbourne
1300 Morris Park Avenue F411                           Royal Parade
Bronx NY 10461 (USA)                                   University of Melbourne Vi 3010 (AUSTRALIA)
Phone: 1-718-430-3766                                  Phone: 61 3 83445164
FAX: 1-718-430-8701                                    FAX: 61 3 83445139
e-mail: pandrade@aecom.yu.edu                          e-mail: alex.a@unimelb.edu.au
Web: http://www.aecom.yu.edu                           Web:
                                                         http://www.genetics.unimelb.edu.au/research/andr
ALCAZAR FUOLI Laura
Department of Microbiology                             ARKOWITZ Robert
Imperial College London                                Institute of Developmental Biology and Cancer
South Kensington Campus                                CNRS UMR6543 - University of Nice-Sophia
London SW7 (UNITED KINGDOM)                            Antipolis
Phone: 004420 7594 5293                                Parc Valrose, Faculté des Sciences
FAX: 00442075943076                                    Nice 06108 (FRANCE)
e-mail: l.alcazar-fuoli@imperial.ac.uk                 Phone: +33 (0)4 92 076425
Web: http://www3.imperial.ac.uk                        FAX: +33 (0)4 92 076466
                                                       e-mail: arkowitz@unice.fr
ALVAREZ Francisco                                      Web:
Cell Biology                                             http://www.unice.fr/isdbc/equipe/equipe.php?id=
Wenner-Gren Institute                                  12
Svante Arrheniusväg 16-18
Stockholm 106 91 (SWEDEN)                              BADER Oliver
Phone: +46 8 16 28 36                                  Institute for Medical Microbiology
FAX: +46 8 15 98 37                                    University Göttingen
e-mail: javier.alvarez@wgi.su.se                       Kreuzbergring 57
Web: http://www.wgi.su.se/                             Göttingen 37075 (GERMANY)
                                                       Phone: +49 (551) 39 22346
AMARSAIKHAN Ansalmaa                                   FAX: +49(551) 39 5861
Biotechnology                                          e-mail: obader@gwdg.de
Middle East Technical University
ODTU                                                   BAHYT Kalieva
Ankara 06531 (TURKEY)                                  Scientific center for drug research "KazBioMed"
Phone: 0090 312 210 56 41                              15 Toraigyrov st., ap. 22
FAX: 0090 312 210 27 67                                Almaty 050043 (KAZAKHSTAN)
e-mail :nansa89@yahoo.com                              Phone: +77051900585
                                                       FAX: +77272206467
ANDREWS Brenda                                         e-mail: space-rover@mail.ru
Centre for Cellular & Biomolecular Research
University of Toronto                                  BASSILANA Martine
Rm 230 160 College Street                              Institute of Developmental Biology and Cancer
Toronto ON M5S 3E1 (CANADA)                            CNRS UMR6543 - University of Nice-Sophia
Phone: 416-978-8562                                    Antipolis
FAX: 416-946-8253                                      Parc Valrose, Faculté des Sciences
e-mail: brenda.andrews@utoronto.ca                     Nice 06108 (France)
Web: http://www.utoronto.ca/andrewslab/                Phone: +33 (0)4 9207 6464
                                                       FAX: +33 (0)4 9207 6466
                                                       e-mail: mbassila@unice.fr
                                                       Web:
                                                         http://www.unice.fr/isdbc/equipe/equipe.php?id=
                                                       12




                                                 193
BAUER Ruth                                              BEZERRA Ana Rita
Medizinische Klinik und Poliklinik II                   CESAM - Department of Biology
Universitaet Wuerzburg                                  University of Aveiro
Josef-Schneider Str. 2                                  Campus Universitário de Santiago
Wuerzburg 97080 (GERMANY)                               Aveiro 3810-193 (PORTUGAL)
Phone: +49 (0)931 201 36408                             Phone: +351 234 370 350 (lab 22754)
FAX: +49 (0)931 201 36409                               FAX: +351 234 372 587
e-mail: bauer_r1@klinik.uni-wuerzburg.de                e-mail: armbezerra@ua.pt
Web: http://www.klinik.uni-
wuerzburg.de/deutsch/home/content.html                  BIGNELL Elaine
                                                        Microbiology
BAUEROVA Vaclava                                        Imperial College London
Institute of Organic Chemistry and Biochemistry         Armstrong Road
Flemingovo n.2                                          London SW7 2AZ (UNITED KINGDOM)
Prague 16610 (CZECH REPUBLIC)                           Phone: 00442075942074
Phone: +420 220 183 242                                 FAX: 00442075943095
FAX: +420 220 183 556                                   e-mail: e.bignell@imperial.ac.uk
e-mail: vaclava@uochb.cas.cz                            Web: http://www1.imperial.ac.uk/medicine/about/d
Web: http://www.iocb.cz                                 ivisions/is/microbiology/aspergillus/

BEN-ZVI Haim (Sharon)                                   BITO Arnold
Department of Human Microbiology and                    Department of Cell Biology
Immunology                                              University of Salzburg
Tel Aviv University                                     Hellbrunnerstrasse 34
Haim Levanon , Ramat-Aviv                               Salzburg 5020 (AUSTRIA)
Tel Aviv 69978 (ISRAEL)                                 Phone: +43 (0)662 8044 5793
e-mail: haimpossible@gmail.com                          FAX: +43 (0)662 8044 144
                                                        e-mail: arnold.bito@sbg.ac.at
BERGMANN Anna
Research Center for Infectious Diseases                 BLATZER Michael
Roentgenring 11                                         Department for Molecularbiology
Wuerzburg 97070 (GERMANY)                               Biozentrum Innsbruck Medical University
Phone: +49-931-31-2125                                  Fritz Pregl Strasse 3
FAX: +49-931-312578                                     Innsbruck A 6020 (AUSTRIA)
e-mail: Anna.Bergmann@uni-wuerzburg.de                  Phone: +43 (0)512 9003 70231
                                                        FAX: +43 (0)512 9003 73100
BERMAN Judith                                           e-mail: Michael.Blatzer@i-med.ac.at
Genetics, Cell Biology and Development
University of Minnesota                                 BOHOVYCH Iryna
6-160 Jackson H, 321 Church St. SE                      School of Medical Sciences
Minneapolis MN 55455 (USA)                              University of Aberdeen
Phone: 612-625-1971                                     Foresterhill
FAX: 612-625-5754                                       Aberdeen AB25 2ZD (UNITED KINGDOM)
e-mail: jberman@umn.edu                                 Phone: +44 1224 555928
Web: http://www.cbs.umn.edu/labs/berman/                FAX: +44 1224 555844
                                                        e-mail: e.k.swaine@abdn.ac.uk
BERTUZZI Margherita
Dept. of Microbiology                                   BONHOMME Julie
Imperial College London                                 Fungal Biology and Pathogenicity
Armstrong Road                                          Institut Pasteur
London UK SW7 2AZ (UNITED KINGDOM)                      25, rue du Docteur Roux
Phone: +44(0)20 7594 5293                               Paris 75015 (FRANCE)
FAX: +44(0)20 7594 3095                                 Phone: +33 (0)1 45 68 82 05
e-mail: margherita.bertuzzi06@imperial.ac.uk            FAX: +33 (0)1 45 68 89 38
                                                        e-mail: julie.bonhomme@pasteur.fr
                                                        Web: http://www.pasteur.fr/




                                                  194
BRACHHOLD Martina                                           CABRAL Vitor
Molecular Biotechnology                                     Fungal Biology and Pathogenicity
Fraunhofer IGB                                              Institut Pasteur
Nobelstrasse 12                                             25 rue du Docteur Roux
Stuttgart 70569 (GERMANY)                                   Paris 75015 (FRANCE)
Phone: +49 (0)711 970 4145                                  Phone: +33 1 40 61 31 26
FAX: +49 (0)711 970 4200                                    FAX: +33 1 45 68 89 38
e-mail: Martina.Brachhold@igb.fraunhofer.de                 e-mail: vitor.cabral@pasteur.fr
Web: www.igb.fraunhofer.de                                  Web: http://www.pasteur.fr/bpf

BRAKHAGE Axel A.                                            CAIRNS Timothy
Molecular and Applied Microbiology                          Department of Microbiology
Leibniz-Institute (HKI), University of Jena                 Imperial College London
Beutenbergstrasse 11a                                       Armstrong Road
Jena 07745 (GERMANY)                                        London SW7 2AZ (UNITED KINGDOM)
Phone: +49 (0)3641 532 1001                                 Phone: 0044 02075945293
FAX: +49 (0)3641 532 0802                                   FAX: 0044 02075943095
e-mail: axel.brakhage@hki-jena.de                           e-mail: t.cairns07@imperial.ac.uk
Web: www.hki-jena.de                                        Web: http://www3.imperial.ac.uk/cmmi

BROWN Alistair JP                                           CANTERO Pilar D.
School of Medical Sciences                                  Institut fuer Mikrobiologie
University of Aberdeen                                      Heinrich Heine Universitaet
Institute of Medical Sciences, Foresterhill                 Universitaetsstrasse 1
Aberdeen AB25 2ZD (UNITED KINGDOM)                          Duesseldorf 40225 (GERMANY)
Phone: +44 (0)1225 555883                                   Phone: +49 211 8114835
FAX: +44 (0)1225 555844                                     FAX: +49 211 8115176
e-mail: al.brown@abdn.ac.uk                                 e-mail: soile@usal.es
Web:
  http://www.abdn.ac.uk/ims/staff/details.php?id=al         CASSONE Antonio
.brown                                                      Infectious Diseases
                                                            Istituto Superiore di Sanità
BRUL Stanley                                                Viale Regina Elena, 299
Molecular Biology & Microbial Food Safety                   Rome 00161 (ITAMY)
SILS University of Amsterdam                                Phone: +390649387113
166 Nieuweachtergracht                                      FAX: +390649387183
Amsterdam NH 1018 WV (NETHERLANDS)                          e-mail: cassone@iss.it
Phone: 31205256970                                          Web: www.iss.it
FAX: 31205256971
e-mail: s.brul@uva.nl                                       CHABRIER-ROSELLO Yeissa
Web: http://home.medewerker.uva.nl/s.brul/                  Pediatrics and Microbiology/Immunology
                                                            University of Rochester School of Medicine &
BUTLER Geraldine                                            Dentistry
School of Biomolecular and Biomedical Science               601 Elmwood Ave
University College Dublin                                   Rochester NY 14642 (USA)
Belfield                                                    Phone: 939-579-4608
Dublin 4 (IRELAND)                                          FAX: 585-273-1104
Phone: +353-1-7166885                                       e-mail: yeissa_chabrierrosello@urmc.rochester.edu
FAX: +353-1-2837211
e-mail: gbutler@ucd.ie                                      CHAKRABORTY Uttara
                                                            Molecular Biology and Genetics Unit
BYRNES III Edmond J.                                        Jawaharlal Nehru Centre for Advanced Scientific
Molecular Genetics and Microbiology                         Research
Duke University                                             Jakkur
312 CARL Building, Research Drive DUMC                      Bangalore 560064 (INDIA)
Durham NC 27713 (USA)                                       Phone: +91 80 2208 2878
Phone: 919-768-3981                                         FAX: +918022082766
FAX: 919-684-5458                                           e-mail: uttara@jncasr.ac.in
e-mail: edmond.byrnes@duke.edu




                                                      195
CHEN Jiangye                                              CORREIA Alexandra
State Key Laboratory of Molecular Biology                 Centro de Biologia Molecular e Ambiental
Institute of Biochemistry and Cell Biology, SIBS,         Universidade do Minho
CAS                                                       Campus de Gualtar
320 Yue Yang Road                                         Braga 4710 057 (PORTUGAL)
Shanghai 200031 (CHINA)                                   Phone: +351 253604310
Phone: 86-21-54921251                                     FAX: +351 253678980
FAX: 86-21-54921011                                       e-mail: alexandracorreia@bio.uminho.pt
e-mail: jychen@sibs.ac.cn                                 Web: http://www.cbma.bio.uminho.pt
Web: http://www.sibs.ac.cn/
                                                          COSTA-DE-OLIVEIRA Sofia
CITIULO Francesco                                         Microbiology
Oral Microbiology                                         Porto Faculty of Medicine
Dublin Dental school and Hospital                         Alameda Prof. Hernani Monteiro
Lincoln place                                             Porto 4200-319 PORTUGAL)
Dublin ABC123 (IRELAND)                                   Phone: +351 225513662
Phone: +353857067837                                      FAX: +351 225513662
FAX: 0035316127295                                        e-mail: sqco@med.up.pt
e-mail: francesco.citiulo@dental.tcd.ie                   Web: http://www.med.up.pt

COADY Alison                                              COSTE Alix
Department of Microbiology and Immunology                 Institute of Microbiology of the University of
University of California, San Francisco                   Lausanne
513 Parnassus Ave, Box 0414                               University of Lausanne,University Hospital Center
San Francisco CA 94143 (USA)                              Bugnon, 48
Phone: +1-415-502-4810                                    Lausanne 1011 (SWITZERLAND)
FAX: +1-415-476-8201                                      Phone: +41 (0)21 314 40 61
e-mail: alison.coady@ucsf.edu                             FAX: +41 (0)21 314 40 60
                                                          e-mail: alix.coste@chuv.ch
COELHO Carolina                                           Web: http://www.chuv.ch/imul
Medical Mycology Yeast Research Group
Center for Neuroscience and Cell Biology                  CURADO Filipa
Rua Larga                                                 Medical Mycology Yeast Research Group
Coimbra 3004-504 (PORTUGAL)                               Center for Neuroscience and Cell Biology
Phone: +351 239 857772                                    Faculdade de Medicina Universidade de Coimbra
FAX: +351 239 822776                                      Coimbra 3004-504 (Portugal)
e-mail: carolinaipcoelho@gmail.com                        Phone: +351 239 857772
                                                          FAX: +351 239 822776
COENYE Tom                                                e-mail: filipa.curado@gmail.com
Lab of Pharmaceutical Microbiology
Ghent University                                          DALLE Frédéric
Harelbekestraat 72                                        Laboratoire Interaction Agents Transmissibles et
Gent 9000 (BELGIUM)                                       muqueuses
Phone: +32(0)92648141                                     Université de Bourgogne
FAX: +32(0)92648195                                       7 boulevard Jeanne d'Arc
e-mail: tom.coenye@ugent.be                               Dijon 21000 (FRANCE)
Web:                                                      Phone: +33 (0)3 80295014
  http://www.ugent.be/fw/en/research/pharmaceutic         FAX: +33 (0)3 80293280
al-analysis/pmicro                                        e-mail: frederic.dalle@u-bourgogne.fr

CORREA-BORDES Jaime                                       DEAK Eszter
Ciencias Biomédicas. Facultad de Ciencias                 Mycotic Diseases Branch
Universidad de Extremadura                                Centers for Disease Control
Avda Elvas sn                                             1600 Clifton Rd.
Badajoz 06071 (SPAIN)                                     Atlanta GA 30333 (USA)
Phone: +34924289300 ext 86874                             Phone: 404-639-1342
FAX: +34924289300                                         FAX: 404-639-3546
e-mail: jcorrea@unex.es                                   e-mail: guf0@cdc.gov




                                                    196
D'ENFERT Christophe                                       EPP Elias
Fungal Biology and Pathogenicity                          Biology
Institut Pasteur                                          McGill University
25 rue du Docteur Roux                                    1205 Docteur Penfield
Paris 75015 (FRANCE)                                      Montréal H3A 1B1 (CANADA)
Phone: +33 1 40 61 32 57                                  Phone: 001 514 496 1529
FAX: +33 1 45 68 89 38                                    FAX: 001 514 496 6213
e-mail: christophe.denfert@pasteur.fr                     e-mail: elias.epp@mail.mcgill.ca
Web: http://www.pasteur.fr/bpf
                                                          FERRANDON Dominique
DERVLA Isaac                                              UPR 9022 du CNRS IBMC
Microbiology and Immunology                               CNRS
University of California - San Francisco                  15, rue R. Descartes
513 Parnassus Ave, Box 0414                               Strasbourg F67084 (FRANCE)
San Francisco CA 94143 (USA)                              Phone: 33 3 88 41 70 17
Phone: 1 415 502 4810                                     FAX: 33 3 88 41 70 17
FAX: 1 415 476 8201                                       e-mail: D.Ferrandon@ibmc.u-strasbg.fr
e-mail: Dervla.Isaac@ucsf.edu
                                                          FERRARI Sélène
DING Chen                                                 Institute of Microbiology
School of Biomolecular and Biomedical Science             University of Lausanne and University Hospital
Conway Institute                                          Center
Belfield                                                  Bugnon 48
Dublin NA (IRELAND)                                       Lausanne 1011 (SWITZERLAND)
Phone: +3531716 6841                                      Phone: +41213144062
FAX: +35312837211                                         FAX: +41213144060
e-mail: chen.ding@ucd.ie                                  e-mail: selene.ferrari@chuv.ch

DISTEL Ben                                                FILLER Scott
Medical Biochemistry                                      Department of Medicine
AMC                                                       Los Angeles Biomedical Research Institute
Meibergdreef 15                                           1124 W. Carson St.
Amsterdam 1105 AZ (NETHERLANDS)                           Torrance CA 90502 (USA)
Phone: +31-20-5665127                                     Phone: 01 310 222-6426
FAX: +31-20-6915519                                       FAX: 01 310 782-2016
e-mail: b.distel@amc.uva.nl                               e-mail: sfiller@ucla.edu

DYER Paul S.                                              FIORI Alessandro
School of Biology                                         VIB Department of Molecular Microbiology
University of Nottingham                                  Katholieke Universiteit Leuven
University Park                                           Kasteelpark Arenberg 31
Nottingham NG7 2RD (UNITED KINGDOM)                       Heverlee 3001 (BELGIUM)
Phone: +44 (0)115 9513203                                 Phone: +32-(0)16320368
FAX: +44 (0)115 9513251                                   FAX: +32 (0)16321979
e-mail: paul.dyer@nottingham.ac.uk                        e-mail: alessandro.fiori@mmbio.vib-kuleuven.be
Web:
  http://www.nottingham.ac.uk/biology/contacts/dy         GABALDON Toni
er/                                                       Bioinformatics and Genomics
ENGEL Jakob                                               Centre for Genomic Regulation (CRG)
Medizinische Hochschule Hannover                          Dr. Aiguader, 88
Zentrum Biochemie                                         Barcelona 08003 (SPAIN)
Abteilung Zelluläre Chemie OE 4330                        Phone: +34 933160281
Carl-Neuberg Str. 1                                       FAX: +34 93 396 99 83
D-30625 Hannover (GERMANY)                                e-mail: tgabaldon@crg.es
Phone: +49 (0)511 532 3367                                Web: www.crg.es/comparative_genomics
FAX: +49 (0)511 532 3956
e-mail : Engel.Jakob@MH-Hannover.de
Web: http://www.mh-hannover.de/




                                                    197
GACSER Attila                                         GILDOR Tsvia
Department of Microbiology                            Molecular Microbiology
University of Szeged                                  Technion
Kozep fasor 52                                        Efron 2
Szeged 6726 (HUNGARY)                                 Haifa 31096 (ISRAEL)
Phone: +36 62 544849                                  Phone: (0) 972 4 8295258
FAX: +36 62 544823                                    FAX: (0) 972 4 8295254
e-mail: gacsera@gmail.com                             E-mail: tsvia@tx.technion.ac.il

GALITSKI Timothy                                      GOLDMAN William
Institute for Systems Biology                         Department of Microbiology and Immunology
1441 N 34th Street                                    The University of North Carolina at Chapel Hill
Seattle WA 98103 (USA)                                116 Manning Drive, Campus Box 7290
Phone: 1 206 732 1206                                 Chapel Hill NC 27599 (USA)
FAX: 1 206 732 1260                                   Phone: +1 919 966 9580
e-mail: tgalitski@systemsbiology.org                  FAX: +1 919 962 8103
                                                      e-mail: goldman@med.unc.edu
GASPARYAN Arsen                                       Web:
Biochemistry                                            http://microimm.med.unc.edu/facultydetail.aspx?i
Yerevan State University                              d=210
A. Manoogian str. 1
Yerevan 0025 (ARMENIA)                                GOMEZ-RAJA Jonathan
Phone: (+374 99) 238686                               Microbiology
FAX: (+374 10) 55-46-41                               University of Minnesota
e-mail: gasparyan.arsen@yahoo.com                     420 Delaware St
                                                      Minneapolis MN 55455 (USA)
GASTEBOIS Amandine                                    Phone: +1 612-624-7994
Parasitology Mycology                                 FAX: +1 (612) 626-0623
Institut Pasteur                                      e-mail: gomez131@umn.edu
25 rue du Docteur Roux
Paris 75015 (France)                                  GONCALVES Teresa
Phone: +33(0)1 45 68 82 25                            Medical Mycology Yeast Research Group
FAX: +33(0)1 40 61 34 19                              Center for Neuroscience and Cell Biology
e-mail: agastebo@pasteur.fr                           Rua Larga
Web: http://www.pasteur.fr/                           Coimbra 3004-504 (PORTUGAL)
                                                      PORTUGAL
GIBSON Amanda                                         Phone : +351 239 857 772
Ecologie, Systématique et Evolution, UMR 8079         FAX: +351 239 822 776
CNRS, ESE                                             e-mail: tmfog@ci.uc.pt
Université de Paris-Sud XI
Bâtiment 360                                          GONZALEZ-NOVO Alberto
Orsay 91405                                           Microbiology and Genetics
FRANCE                                                CSIC/ University of Salamanca
Phone: +33 (0) 6 7157 2710                            Campus Unamuno
FAX: +33 (0) 1 6915 4697                              Salamanca 37007 (SPAIN)
e-mail: Amanda.Gibson@u-psud.fr                       Phone: +34 923 294462
                                                      FAX: +34 923 224876
GIEFING Carmen                                        e-mail: anovo@usal.es
Molecular Microbiology                                Web: http://www.imb.usal-csic.es/
Intercell AG
Campus Vienna Biocenter 3                             GRUMBT Maria
Vienna 1030 (AUSTRIA)                                 Fundamental Molecular Biology of Pathogenic
Phone: +43-1-20620                                    Fungi, Hans Knoell Institute
FAX: +43-1-20620-801                                  Beutenbergstr. 11a
e-mail: cgiefing@intercell.com                        Jena 07745 (GERMANY)
Web: www.intercell.com                                Phone: +49 3641 532 1247
                                                      FAX: +49 3641 532 0809
                                                      e-mail: maria.grumbt@hki-jena.de
                                                      Web: www.hki-jena.de




                                                198
GUIDA Alessandro                                     HEITMAN Joseph
School of Medical science                            Molecular Genetics and Microbiology
Conway Institute - UCD                               Duke University Medical Center
Belfield                                             Research Drive, 322 CARL Building, Box 3546
Dublin 04 (IRELAND)                                  Durham NC 27710 (USA)
Phone: +353 (0) 871337884                            Phone: 919 684-2824
FAX: none                                            FAX: 919 684-5458
e-mail: alessandro.guida@ucd.ie                      e-mail: heitm001@duke.edu
                                                     Web:
GUTIERREZ-ESCRIBANO Pilar                              http://www.mgm.duke.edu/microbial/mycology/h
Ciencias Biomédicas. Facultad de Ciencias            eitman/
Universidad de Extremadura
Avda Elvas sn                                        HILLER Ekkehard
Badajoz 06071 (SPAIN)                                Universität Stuttgart
Phone: +34924289300 ext 86874                        Nobelstrasse 12
FAX: +34924289300                                    Stuttgart 70569 (GERMANY)
e-mail: pilargutierrez@unex.es                       Phone: +49 (0)711 970 4171
                                                     FAX: +49 (0)711 970 4200
HALL Rebecca                                         e-mail: ekkehard.hiller@igvt.uni-stuttgart.de
Department of Biosciences                            HÖFER Thomas
University of Kent                                   Modeling of Biological Systems
Giles Lane                                           German Cancer Research Center
Canterbury CT2 7NJ (UNITED KINGDOM)                  Im Neuenheimer Feld 280
Phone: +44 (0) 1227 823735                           Heidelberg 69120 (GERMANY)
FAX: +44 (0)1227 763912                              Phone: 004962215451380
e-mail: r.a.hall@kent.ac.uk                          FAX: 004962215451487
Web: http://www.kent.ac.uk/bio/muhlschlegel/         e-mail: t.hoefer@dkfz-heidelberg.de
                                                     Web: www.dkfz.de
HAYNES Ken
Microbiology                                         HOLCOMBE Lucy
Imperial College London                              Department of Microbiology
The Flowers Building                                 University College Cork
London SW7 2AZ (UNITED KINGDOM)                      College Road
Phone: +44 (0)20 7594 2072                           Cork City Co. Cork (IRELAND)
FAX: +44 (0)20 7594 3095                             Phone: +353 (0)21 4902934
e-mail: k.haynes@imperial.ac.uk                      FAX: +353 (0)21 4903101
                                                     e-mail: l.holcombe@ucc.ie
HEILMANN Clemens J.                                  Web:
Massspectrometry of Biomacromolecules                  http://www.ucc.ie/en/tramways/research/Fungal/
Swammerdam Institute for Life Sciences
Nieuwe Achtergracht 166                              HRUSKOVA-HEIDINGSFELDOVA Olga
Amsterdam 1018 WV                                    Gilead Sciences Research Centre
THE NETHERLANDS                                      Institute of Organic Chemistry and Biochemistry
Phone: +31 (0)20 525 5669                            Flemingovo nam. 2
FAX: +31 (0)20 525 6971                              Prague 6 166 10 (CZECH REPUBLIC)
e-mail: clemens.j.heilmann@gmail.com                 Phone: +420 220 183 249
                                                     FAX: +420 224 310 090
HEINEKAMP Thorsten                                   e-mail: olga-hh@uochb.cas.cz
Molecular and Applied Microbiology                   Web: www.uochb.cas.cz
Hans Knoell Institute
Beutenbergstr. 11a                                   HSUEH-LUI Ho
Jena 07745 (GERMANY)                                 Department of Microbiology
Phone: +49 (0)3641 532 1095                          Imperial College London
FAX: +49 (0)3641 532 0803                            5.40 Armstrong Road
e-mail: thorsten.heinekamp@hki-jena.de               London SW7 2AZ (UNITED KINGDOM)
Web: http://www.hki-jena.de                          Phone: +44 02075947409
                                                     FAX: +44 02075943095
                                                     e-mail: hsueh-lui.ho04@imperial.ac.uk
                                                     Web: http://www3.imperial.ac.uk/cmmi




                                               199
HUBE Bernhard                                             JONES Laura
Microbial Pathogenicity Mechanisms                        Molecular Biology and Biotecnology
Leibniz Institute for Nat. Prod. Res. and Inf.            University of Sheffield
Biology e. V.                                             Western Bank
Beutenbergstraße 11a                                      Sheffield S10 2TN (UNITED KINGDOM)
Jena Th 07745 (GERMANY)                                   Phone: +44(0)1142222748
Phone: +49 (0) 3641 532 1401                              FAX: +44(0)1142222748
FAX: +49 (0) 3641 532 0810                                e-mail: mbp05laj@sheffield.ac.uk
e-mail: bernhard.hube@hki-jena.de                         Web: www.shef.ac.uk
Web: www.hki-jena.de
                                                          JÜRGENSEN Claudia
IDNURM Alexander                                          School of Biomolecular and Biomedical Science
School of Biological Sciences                             University College Dublin
University of Missouri-Kansas City                        Conway Institute
5100 Rockhill Road                                        Belfield Dublin 4 (IRELAND)
Kansas City MO 64110 (USA)                                Phone: +353 1 716 6838
Phone: +1 816 235 2265                                    FAX: +353 1 283 7211
FAX: +1 816 235 1503                                      e-mail: claudia.jurgensen@ucd.ie
e-mail: idnurma@umkc.edu                                  Web: www.ucd.ie
Web:
  http://sbs.umkc.edu/people/faculty/docIdnurmA.c         KEELING Patrick
fm                                                        Botany
                                                          University of British Columbia
IFRIM Daniela                                             6270 University Blvd.
INRA AGRO PARIS TECH                                      Vancouver BC V6T 1Z4
Avenue Brétignières                                       CANADACanada
Résidence Jacques Ratineau, chambre # 110                 Phone: 604 8224906
78850 Thiverval-Grignon                                   FAX: 604 8226089
                                                          e-mail: pkeeling@interchange.ubc.ca
ISAAC Dervla                                              Web: www.botany.ubc.ca/keeling
Microbiology and Immunology
University of California - San Francisco                  KENIYA Mikhail
513 Parnassus Ave, Box 0414                               Oral Sciences
San Francisco CA 94143 (USA)                              University of Otago
Phone: 1 415 502 4810                                     310 Great King Street
FAX: 1 415 476 8201                                       Dunedin 9016 (NEW ZEALAND)
e-mail: Dervla.Isaac@ucsf.edu                             Phone: +64 (3) 479 3873
                                                          FAX: +64 (3) 479 7078
JACOBSEN lse D.                                           e-mail: mikhail.keniya@otago.ac.nz
Microbial Pathogenicity Mechanisms
Hans Knoell Institute                                     KLIS Frans
Beutenbergstra§e 11a                                      Swammerdam Inst Life Sciences
Jena 07745 (GERMANY)                                      University of Amsterdam
Phone: +49 (0) 3641 532 1223                              Nieuwe Achtergracht 166
FAX: +49 (0) 3641 532 0810                                Amsterdam 1018WV (NETHERLANDS)
e-mail: ilse.jacobsen@hki-jena.de                         Phone: +31-20-525 7834
Web: http://www.hki-jena.de/index.php                     FAX: +31-20-525 7924
                                                          e-mail: F.M.Klis@uva.nl
JANBON Guilhem                                            Web: http://home.medewerker.uva.nl/f.m.klis/
Unité des Aspergillus
Institut Pasteur                                          KORNITZER Daniel
25 rue du Dr Roux                                         Molecular Microbiology
Paris 75015 (France)                                      Technion Faculty of Medicine
Phone: 33 (0)145688356                                    2, Efron St.
FAX: 33 (0)145688420                                      Haifa 31096 (ISRAEL)
e-mail: janbon@pasteur.fr                                 Phone: +972 (0)4 829 5258
                                                          FAX: +972 (0)4 829 5254
                                                          e-mail: danielk@tx.technion.ac.il




                                                    200
KRAIDLOVA Lucie                                              LEGRAND Mélanie
Department of Membrane Transport                             Fungal Biology and Pathogenicity
Institute of Physiology                                      Institut Pasteur
Videnska 1083                                                25 rue du Docteur Roux
Prague CR 142 20 (CZECH REPUBLIC)                            Paris 75015 (FRANCE)
Phone: +420 777 856 658                                      Phone: +33 (0)1 4061 3126
FAX: +420 296 442 194                                        FAX: +33 (0)1 4568 8938
e-mail: kraidlova@biomed.cas.cz                              e-mail: mlegrand@pasteur.fr
                                                             Web: http://www.pasteur.fr/bpf
KRAUKE Yannick
Dept. Membrane Transport                                     LEMUTH Karin
Institute of Physiology AS CR, v.v.i.                        Molecular Biotechnology
Videnska 1083                                                Fraunhofer IGB
Prague 142 20 (CZECH REPUBLIC)                               Nobelstr. 12
Phone: +420241062120                                         Stuttgart 70569 (GERMANY)
FAX: +420296442488                                           Phone: +497119704044
e-mail: krauke@biomed.cas.cz                                 FAX: +497119704200
                                                             e-mail: karin.lemuth@igb.fraunhofer.de
KUCHLER Karl
Christian Doppler Laboratory for Infection Biology           LESIAK Iwona
Medical University Vienna                                    Molecular Genetic
Dr. Bohr-Gasse 9/2                                           Medical Biochemistry
Vienna A-1030 (AUSTRIA)                                      Dr. Bohr-Gasse 9
Phone: +43-1-4277-61807                                      Vienna 1030 (AUSTRIA)
FAX: +43-1-4277-9618                                         Phone: +43 (0) 4277 61812
e-mail: karl.kuchler@meduniwien.ac.at                        FAX: +43 (0) 4277 9618
                                                             e-mail: iwona.lesiak@univie.ac.at
LA FLEUR Michael                                             Web:
Antimicrobial Discovery Center                                 www.meduniwien.ac.at/medbch/MolGen/kuchler/
Northeastern University
360 Huntington Avenue                                        LINDEMANN Elena
Boston MA 02115 (USA)                                        Molecular Biotechnology
Phone: 1-617-373-5013                                        Fraunhofer IGB
FAX: 1-617-373-3724                                          Nobelstr.12
e-mail: lafleur.m@neu.edu                                    Stuttgart 70569 (GERMANY)
Web: http://www.northeastern.edu                             Phone: +49 (0)711 970 4145
                                                             FAX: +49 (0)711 970 4200
LATGE Jean-Paul                                              e-mail: lind@igb.fraunhofer.de
Parasitology and Mycology                                    Web: www.igb.fraunhofer.de
Institut Pasteur
25 rue du Docteur Roux                                       L'OLLIVIER Coralie
Paris 75015 (FRANCE)                                         laboratoire LIMA (EA562)
Phone: +33 (0)1 40 61 35 18                                  Université de médecine Dijon France
FAX: +33 (0)1 40 61 341 9                                    2 bd du Maréchal de Lattre de Tassigny
e-mail: jean-paul.latge@pasteur.fr                           Dijon 21000 (FRANCE)
Web:                                                         Phone: +33 (0)3 8029 3780
  http://www.pasteur.fr/recherche/unites/aspergillus         FAX: +33 (0)3 8029 3627
/th1-aspergillus.htm                                         e-mail: coralie.lollivier@chu-dijon.fr

LEACH Michelle                                               LORENZ Michael
Aberdeen Fungal Group                                        Microbiology and Molecular Genetics
University of Aberdeen                                       The University of Texas Health Science Center
Institute of Medical Sciences, Foresterhill                  6431 Fannin
Aberdeen AB25 2ZD (UNITED KINGDOM)                           Houston TX 77030 (USA)
Phone: +44 (0)1224 555883                                    Phone: +1 (713) 500-7422
FAX: +44 (0)1224 555844                                      FAX: +1 (713) 500-5499
e-mail: michelle.leach@abdn.ac.uk                            e-mail: Michael.Lorenz@uth.tmc.edu
                                                             Web: http://www.lorenzlab.org




                                                       201
LYNCH Denise B.                                           MENSHIK Bahyt
School of Biomolecular and Biomedical Science             Pharmacology and biochemistry
University College Dublin                                 Scientific center for drug research "KazBioMed"
Donneybrook                                               15 Toraigyrov st., ap. 22
Dublin 04 (IRELAND)                                       Almaty 050043 (KAZAKHSTAN)
Phone: +353 1 716 6838                                    Phone: +77051900585
FAX: +353 1 716 6701                                      FAX: +77272206467
e-mail: denise.lynch@ucd.ie                               e-mail: space-rover@mail.ru

MAI Michaela K.                                           MIRAMON MARTINEZ Pedro
MBT                                                       Microbial Pathogenicity Mechanisms
IGVT, Universität Stuttgart                               Hans Knoell Institute
Nobelstr. 12                                              Beutenbergstrasse 11a
Stuttgart 70569 (GERMANY)                                 Jena 07745 (GERMANY)
Phone: +49 711 970 4171                                   Phone: +49 (0) 3641 532 0359
FAX: +49 711 970 4200                                     FAX: +49 (0) 3641 532 0810
e-mail: michaela.mai@igb.fhg.de                           e-mail: pedro.miramon@hki-jena.de
                                                          Web: http://www.hki-jena.de/index.php
MAJER Olivia
Medical Biochemistry                                      MITCHELL Aaron
Max F. Perutz Laboratories; Medical University of         Biological Sciences
Vienna                                                    Carnegie Mellon University
Dr. Bohrgasse 9/2                                         4400 Fifth Avenue
Vienna 1030 (AUSTRIA)                                     Pittsburgh PA 15213
Phone: +43 1 4277 61812                                   USA
FAX: +43 1 4277 9618                                      Phone: 412-268-5844
e-mail: olivia.majer@meduniwien.ac.at                     FAX: 412-268-7129
Web: www.mfpl.ac.at                                       e-mail: apm1@andrew.cmu.edu
                                                          Web:
MANOHARLAL Raman                                            http://www.cmu.edu/bio/faculty/mitchell.shtml
Membrane Biology Laboratory (MBL)
Jawaharlal Nehru University (JNU)                         MOGENSEN Estelle
School of Life Sciences (SLS)                             Unité des Aspergillus
New Delhi 110067 (INDIA)                                  Institut Pasteur
Phone: +91-11-26704509,+91-9871738536                     25 rue du Docteur Roux
FAX: + 91-11-26741081                                     Paris 75015 (France)
e-mail: ramanbiotech@gmail.com                            Phone: +33 1 45 68 83 56
                                                          FAX: +33 1 45 68 84 20
MARCET-HOUBEN Marina                                      e-mail: estelle.mogensen@pasteur.fr
Bioinformatics and Genomics department                    Web: http://www.pasteur.fr/
CRG (Center for Genomic Regulation)
Doctor Aiguader, 88                                       MORAES NICOLA André
Barcelona 08003 (SPAIN)                                   Microbiology and Immunology
Phone: +34 93 316 02 82                                   Albert Einstein College of Medicine
FAX: +34 93 316 00 99                                     1300 Morris Park avenue
e-mail: mmarcet@crg.es                                    Bronx NY 10461 (USA)
Web: http://www.crg.es/comparative_genomics               Phone: +1-718-430-3766
                                                          FAX: +1-718-430-8701
MELO Nadja                                                e-mail: anicola@aecom.yu.edu
Immunology
Institute of Life Science, Swansea University
Singleton Park
Swansea SA2 8PP (UNITED KINGDOM)
Phone: +44 01792 368578
FAX: +44 01792 301022
e-mail: nadjarm@yahoo.com




                                                    202
MORSCHHÄUSER Joachim                                        NEGRI Melyssa
Institut für Molekulare Infektionsbiologie                  Biological Engineering
Universität Würzburg                                        University of Minho
Röntgenring 11                                              Campus Gualtar
Würzburg D-97070 (GERMANY)                                  Braga 4710-057 PORTUGAL)
Phone: +49 (0)931 312152                                    Phone: +351 253604400
FAX: +49 (0)931 312578                                      FAX: +351 253678986
e-mail: joachim.morschhaeuser@mail.uni-                     e-mail: melyssanegri@deb.uminho.pt
wuerzburg.de                                                Web: http://www.ceb.uminho.pt/biofilm/
Web: http://www.infektionsforschung.uni-
wuerzburg.de/research/mycology_unit/                        NESSEIR Audrey
                                                            Fungal Biology and Pathogenicity
MUHLSCHLEGEL Fritz                                          Institut Pasteur
Biosciences                                                 25 rue Docteur Roux
University of Kent                                          Paris 75015 (FRANCE)
Giles lane                                                  Phone: +33 (0) 1 4061 3126
Canterbury CT27NJ (UNITED KINGDOM)                          FAX: +33 (0) 1 4568 8938
Phone: +44 (0)1227 823988                                   e-mail: audrey.nesseir@pasteur.fr
FAX: +44 (0)1227 763912                                     Web: http://www.pasteur.fr/bpf
e-mail: F.A.Muhlschlegel@kent.ac.uk
Web: http://www.kent.ac.uk/bio/kfg/index.html               NETEA Mihai
                                                            Radboud University Nijmegen Medical Center
MUNRO Carol                                                 Geert Grooteplein 8
School of Medical Sciences                                  Nijmegen 6500 HB (NETHERLANDS)
University of Aberdeen                                      Phone: +31 (0)24 3614652
Institute of Medical Sciences, Foresterhill                 FAX: +31 (0)24 3541734
Aberdeen AB25 2ZD UNITED KINGDOM)                           e-mail: m.netea@aig.umcn.nl
Phone: +44 (0)1224 555927
FAX: +44 (0)1224 555844                                     ODDS Frank
e-mail: c.a.munro@abdn.ac.uk                                Aberdeen Fungal Group
Web:                                                        Institute of Medical Sciences
  http://www.abdn.ac.uk/ims/staff/details.php?id=c.         Foresterhill
a.munro                                                     Aberdeen AB25 2ZD (UNITED KINGDOM)
                                                            Phone: +44 (0) 1224 555828
NAGLIK Julian                                               FAX: +44 no fax no. use email
Oral Immunology                                             e-mail: f.odds@abdn.ac.uk
King's College London
St Thomas Street                                            O' KEEFFE Grainne
London SE1 9RT (UNITED KINGDOM)                             Biology and National Institute for Cellular
Phone: +44 20 7188 4377                                     Biotechnology
FAX: +44 20 7188 4375                                       National University of Ireland Maynooth
e-mail: julian.naglik@kcl.ac.uk                             Maynooth
                                                            Co. Kildare 00000 (IRELAND)
NANTEL André                                                Phone: +353 (0)1 708 3140
Biotechnology Research Institute                            FAX: +353 (0)1 708 3845
National Research Council of Canada                         e-mail: grainneokeeffe@gmail.com
6100 Royalmount
Montreal QC H4P 2R2 (CANADA)                                O'GORMAN Céline M.
Phone: +1 (514) 496-6370                                    UCD School of Biology & Environmental Science
FAX: +1 (514) 496-9127                                      University College Dublin
e-mail: andre.nantel@nrc-cnrc.gc.ca                         Science Centre (West)
                                                            Belfield, Dublin 4 (IRELAND)
                                                            Phone: +353 (0)1 716 2350
                                                            FAX: +353 (0)1 716 1153
                                                            e-mail: celine.ogorman@ucd.ie




                                                      203
OSHEROV Nir                                            QUINTIN Jessica
Human Microbiology                                     Réponse immunitaire et développement chez les
Tel-Aviv University                                    Insectes
Ramat-Aviv                                             Institut de Biologie Moléculaire et Cellulaire
Tel-Aviv 69978 (ISRAEL)                                15 rue René Descartes
Phone: 972 3 640 9599                                  Strasbourg 67084 (FRANCE)
FAX: 972 3 640 9160                                    Phone: + 33 (0) 3 88 41 70 39
e-mail: nosherov@post.tau.ac.il                        FAX: + 33 (0) 3 88 60 69 22
                                                       e-mail: j.quintin@ibmc.u-strasbg.fr
PANWAR Sneh Lata                                       Web: http://www-ibmc.u-strasbg.fr
Jawamarlal Nehru University
Lab n° 106                                             RAMIREZ-ZAVALA Bernardo
School of Life Sciences                                Institut für Molekulare Infektionsbiologie
New Mehrauli Road                                      Universität Würzburg
110067 New Delhi (INDIA)                               Röntgenring 11
Phone : 91 21 26704620                                 Würzburg 97070 (GERMANY)
FAX : 91 11 2674 2558                                  Phone: +0049 931 312127
e-mail : sneh@mail.jnu.ac.in                           FAX: +0049 931 312578
                                                       e-mail: b.ramirez@uni-wuerzburg.de
PRASAD Rajentra
Jawaharlal Nehru University                            RICARDO Elisabete
Membrane Biology Laboratory                            Microbiology
School of Life Sciences                                Faculty of Medicine
New Mehrauli Road                                      Alameda Prof Hernani Monteiro
New Delhi 110067 (INDIA)                               Porto 4200-319 PORTUGAL)
Phone: +91-11-26704509                                 Phone: +351 225513662
FAX: +91-11-26741081                                   FAX: +351 225513662
E-mail : rp47jnu@gmail.com                             e-mail: betaricardo@yahoo.com

PRASAD Tulika                                          ROEMER Terry
Advanced Instrumentation Facility                      Infectious Disease
University Science Instrumentation Centre              Merck & Co., Inc.
Jawaharlal Nehru University                            126 East Lincoln Ave.
New Delhi 110067 (INDIA)                               Rahway NJ 07065 (USA)
Phone: +91-11-26704560                                 Phone: 1-732-594-4906
FAX: +91-11-26741081                                   FAX: 1-732-594-6708
E-mail: prasadtulika@hotmail.com                       e-mail: terry_roemer@merck.com
Web:
  www.geocities.com/ResearchTriangle/lab/5540/         ROSSIGNOL Tristan
                                                       Unité Biologie et Pathogénicité Fongiques
PRUSTY RAO Reeta                                       Institut Pasteur
Biology & Biotechnology                                25, rue du Docteur Roux
WPI                                                    Paris 75015 (FRANCE)
100 Institute Road                                     Phone: +33(0)145688205
Worcester MA 01609 (USA)                               FAX: +33 (0)1 45 68 89 38
Phone: 001-508-831-6120                                e-mail: tristan.rossignol@pasteur.fr
FAX: 001-508-831-5936                                  Web: http://www.pasteur.fr/bpf
e-mail: rpr@wpi.edu
Web: http://users.wpi.edu/~prustyraolab/               ROWAN Raymond
                                                       Oral Microbiology
PURI Nidhi                                             Dublin Dental School & Hospital
Membrane Biology Laboratory (MBL)                      Lincoln Place
Jawaharlal Nehru University (JNU)                      Dublin ABC1234 (IRELAND)
School of Life Sciences (SLS)                          Phone: 00353 85 7735908
New Delhi 110067 (INDIA)                               FAX: 0035316711255
Phone: +91-11-26704509,+91-9810974589                  e-mail: raymond.rowan@dental.tcd.ie
FAX: + 91-11-26741081
e-mail: npuri79@gmail.com




                                                 204
RUPP Steffen                                      SCHEYNIUS Annika
Molecular Biotechnology                           Dept of Medicine Solna
Fraunhofer IGB                                    Karolinska Institutet
Nobelstr. 12                                      L2:04
Stuttgart 70569 (GERMANY)                         Stockholm 171 77 (SWEDEN)
Phone: +49 (0)711 970 4045                        Phone: +46 8 5177 5934
FAX: +49 (0)711 970 4200                          FAX: +46 8 335724
e-mail: Steffen.Rupp@igb.fraunhofer.de            e-mail: annika.scheynius@ki.se
Web: http://www.igb.fraunhofer.de
                                                  SCHINDLER Susann
SADHALE Parag                                     Infection Biology
Microbiology and Cell Biology                     Leibniz Institut -Hans Knöll Institut
Indian Institute of Science                       Beutenbergstrasse 11a
C V Raman Road                                    Jena 07745 (GERMANY)
Bangalore KA 560012 (INDIA)                       Phone: 036415321168
Phone: +91 80 22932292                            FAX: 036415320807
FAX: +91 80 23602697                              e-mail: susann.schindler@hki-jena.de
E-mail: parag.sadhale@gmail.com                   Web: www.hki-jena.de

SANGLARD Dominique                                SCHMAUCH Christian
Inst. of Microbiology                             Institute of Developmental Biology and Cancer,
Univ of Lausanne and Univ Hospital Center         CNRS UMR6543
Bugnon 48                                         Université Nice - Sophia Antipolis
Lausanne 1011 (SWITZERLAND)                       Parc Valrose
Phone: +41 21 3144083                             Nice 06108 (France)
FAX: +41 21 3144060                               Phone: +33 (0)4 9207 6465
e-mail: Dominique.Sanglard@chuv.ch                FAX: +33 (0)4 9207 6466
Web: http://www.chuv.ch/imul/                     e-mail: schmauch@unice.fr
                                                  Web: www.unice.fr/isdbc/
SANTOS Manuel A. S.
CESAM - Department of Biology                     SCHRÖPPEL Klaus
University of Aveiro                              Medical Microbiology and Hygiene
Campus Universitário Santiago                     University of Tübingen
Aveiro 3810-193 PORTUGAL)                         Elfriede-Aulhorn-Str. 6
Phone: +351234370771                              Tübingen 72076 (GERMANY)
FAX: +351 234 372 587                             Phone: +49 (0)7071 29 82358
e-mail: msantos@ua.pt                             FAX: +49 (0)7071 29 5440
Web: http://www.ua.pt/ii/rnomics/                 e-mail: klaus.schroeppel@med.uni-tuebingen.de

SANYAL Kaustuv                                    SCHUBERT Sabrina
Molecular Biology & Genetics                      Institut für molekulare Infektionsbiologie
JNCASR                                            Universität Würzburg
Jakkur                                            Röntgenring 11
Bangalore 560064 (INDIA)                          Würzburg 97070 (GERMANY)
Phone: +91 80 2208 2878                           Phone: +49 931 31 2127
FAX: +91 80 2208 2766                             FAX: +49 931 31 2578
E-mail: sanyal@jncasr.ac.in                       e-mail: s.schubert@uni-wuerzburg.de
Web: http://www.jncasr.ac.in/sanyal               Web: http://www.infektionsforschung.uni-
                                                  wuerzburg.de/
SARAZIN Aurore
Inserm U799-Mycologie Fondamentale et             SCHULLER Christoph
Appliquée                                         Department of Biochemistry
Faculté de médecine, pole recherche               University of Vienna, MFPL
Place Verdun, Avenue Oscar Lambret                Dr.Bohrgasse 9/5
Lille 59045 (FRANCE)                              Vienna A-1030 (AUSTRIA)
Phone: +33 (0) 3 20 62 34 15                      Phone: +43 1 4277 52815
FAX: + 33 (0) 3 20 62 34 16                       FAX: +43 1 4277 9528
e-mail: aurore.sarazin@laposte.net                e-mail: christoph.schueller@univie.ac.at
                                                  Web: http://www.mfpl.ac.at/index.php?cid=81




                                            205
SCHWARZMÜLLER Tobias                                   SIL Anita
Christian Doppler Laboratory, Max F. Perutz            Microbiology and Immunology
Laboratories                                           University of California San Francisco
Medical University of Vienna                           513 Parnassus, S469
Dr. Bohr-Gasse 9/2                                     San Francisco CA 94143-0414 (USA)
Vienna 1030 (AUSTRIA)                                  Phone: 001-415-502-1805
Phone: 00431-4277-61818                                FAX: 001-415-476-8201
FAX: 00431-4277-9618                                   e-mail: sil@cgl.ucsf.edu
e-mail: Tobias.Schwarzmueller@meduniwien.ac.at         Web: http://histo.ucsf.edu/sillab/index.htm
Web:
  http://www.meduniwien.ac.at/medbch/MolGen/k          SILVA Ana
uchler/                                                Department of Microbiology
                                                       Medicine Faculty, University of Porto
SHAHANA Shahida                                        Al. Prof. Hernâni Monteiro
Aberdeen fungal group                                  Porto 4200-319 (PORTUGAL)
Institution of Medical Sciences                        Phone: 00351917123337
Foresterhill                                           FAX: 00351225513662
Aberdeen Ab25 2ZD (UNITED KINGDOM)                     e-mail: anatpsilva@hotmail.com
Phone: +44(0)1224 555878                               Web: www.fmup.pt
FAX: +44(0)1224 555844
e-mail: s.shahana@abdn.ac.uk                           SIMÕES João
                                                       Biologia
SHARMA Monika                                          Universidade de Aveiro
Membrane Biology Laboratory (MBL)                      Campus universitário de santiago
Jawaharlal Nehru University (JNU)                      Aveiro 3810-193 (PORTUGAL)
School of Life Sciences (SLS)                          Phone: +351 234 372 587
New Delhi 110067 (INDIA)                               FAX: +351 234 370 350
Phone: +91-11-26704509,+91-9958411024                  e-mail: joaosimoes@ua.pt
FAX: + 91-11-26741081                                  Web: http://www.ua.pt/ii/rnomics
e-mail: monikabiotech@gmail.com
                                                       SIXIANG Sai
SHEKHOVTSOVA Elena                                     School of Biomolecular and Biomedical Science
Immunology                                             Conway Institute
Shemyakin and Ovchinnikov Institute of                 University College Dublin, Belfield
Bioorganic Chemistry                                   Dublin 4 (IRELAND)
Miklukho-Maklaya st., 16/10                            Phone: +353879454870
Moscow 117997 (RUSSIA)                                 FAX: +353-1-2837211
Phone: +7 495 330 40 11                                e-mail: sixiang.sai@ucd.ie
FAX: +7 495 330 40 11                                  Web: http://www.ucd.ie/biochem/gb/Lab/
e-mail: shehovcova_elena@mail.ru
                                                       SOHN Kai
SHEVCHENKO Marina                                      MBT
Immunology                                             Fraunhofer IGB
Shemyakin-Ovchinnikov Institute of Bioorganic          Nobelstr. 12
Chemistry                                              Stuttgart 70569 (GERMANY)
Miklukho-Maklaya 16/10                                 Phone: +49 (0)711 970 4055
Moscow 117997 (RUSSIA)                                 FAX: +49 (0)711 970 4200
Phone: +74953304011                                    e-mail: kso@igb.fhg.de
FAX: +74953304011
e-mail: shev@mx.ibch.ru                                SORGO Alice
Web: http://www.ibch.ru/                               Swammerdam Institute for Life Sciences
                                                       University of Amsterdam
                                                       Nieuwe Achtergracht 166
                                                       Amsterdam 1018 WV (NETHERLANDS)
                                                       Phone: +31 (0) 6 5757 4190
                                                       FAX: +31 (0) 20525 7924
                                                       e-mail: a.g.sorgo@uva.nl




                                                 206
SOYLER Betul                                              SULLIVAN Derek
Fungal Molecular Genetics and Enzymology                  School of Dental Science
METU                                                      Trinity College Dublin
ODTU Gida Muhendisligi Bolumu                             Lincoln Place
Ankara – 06531 (TURKEY)                                   Dublin 2 (IRELAND)
Phone: +903122105641                                      Phone: +353 (0)1 612 7275
FAX: +903122102767                                        FAX: +353 (0)1 612 7295
e-mail: betulsoyler06@gmail.com                           e-mail: Derek.Sullivan@dental.tcd.ie
                                                          Web: http://people.tcd.ie/djsullvn
STAIB Peter
Fundamental Molecular Biology of Pathogenic               SYCHROVA Hana
fungi                                                     Membrane Transport
Hans-Knoell-Institute                                     Institute of Physiology, AS CR
Beutenbergstr. 11a                                        Videnska 1083
Jena 07745 (GERMANY)                                      Prague 4 14220 (CZECH REPUBLIC)
Phone: +49 (0) 3641 532 1600                              Phone: 420-241062667
FAX: +49 (0) 3641 532 0809                                FAX: 420-241062488
e-mail: peter.staib@hki-jena.de                           e-mail: sychrova@biomed.cas.cz
Web: www.hki-jena.de                                      Web: http://sun2.biomed.cas.cz/fgu/en/index.

STEVENS Rebecca                                           SYNNOTT John
MBT                                                       UCD School of Biomolecular and Biomedical
Fraunhofer IGB                                            Science
Nobelstrasse 12                                           Conway Institute, University College Dublin
Stuttgart 70569 (GERMANY)                                 Belfield
Phone: +49 (0)711-970-4048                                Dublin D4 (IRELAND)
FAX: +49 (0)711-970-4200                                  Phone: +353 (0)1 716 6838
e-mail: Rebecca.Stevens@igb.fraunhofer.de                 FAX: +353 (0)1 716 6701
Web: www.igb.fraunhofer.de                                e-mail: john.synnott@ucd.ie
                                                          Web: http://www.ucd.ie/biochem/gb/Lab/
STICHTERNOTH Catrin
Institut fuer Mikrobiologie                               TALBOT Nicholas
Heinrich Heine Universitaet                               Biosciences
Universitaetsstr. 1                                       University of Exeter
Duesseldorf 40225 (GERMANY)                               Geoffrey Pope Building
Phone: +49 211 8114835                                    Exeter EX4 4QD (UNITED KINGDOM)
FAX: +49 211 8115176                                      Phone: +44 1392 269151
e-mail: C.Stichternoth@uni-duesseldorf.de                 FAX: +44 1392 263434
                                                          e-mail: n.j.talbot@exeter.ac.uk
SU Chang                                                  Web: http://cogeme.ex.ac.uk/talbot
State Key Laboratory of Molecular Biology
Institute of Biochemistry and Cell Biology, SIBS,         TAYLOR John W.
CAS                                                       Plant and Microbial Biology
320 Yue Yang Road                                         University of California, Berkeley
Shanghai 200031 (CHINA)                                   111 Koshland Hall
Phone: 86-21-54921152                                     Berkeley CA 94720-3102 (USA)
FAX: 86-21-54921011                                       Phone: + 510 642 5366
e-mail: csu@sibs.ac.cn                                    FAX: + 510 642 4995
Web: http://www.sibs.ac.cn/                               e-mail: jtaylor@nature.berkeley.edu
                                                          Web: http://pmb.berkeley.edu/~taylor/
SUDBERY Peter
Molecular Biology and Biotechnology
Sheffield University
Western Bank
Sheffield S10 2TN (UNITED KINGDOM)
Phone: +44 114 2226186
FAX: +44 114 2222800
e-mail: P.Sudbery@shef.ac.uk




                                                    207
TIERNEY Lanay                                          VAN DIJCK Patrick
Department of Medical Biochemistry                     Molecular Microbiology, Laboratory of Molecular
Max F. Perutz Laboratories                             Cell Biology
Dr. Bohr-Gasse 9/2                                     VIB, K.U. Leuven
Vienna 1030 (AUSTRIA)                                  Kasteelpark Arenberg 31
Phone: +43 (0)1 4277 6181 2                            Leuven B-3001 (BELGIUM)
FAX: +43 (0)1 4277 9618                                Phone: +32(0)16 321512
e-mail: Lanay.Tierney@meduniwien.ac.at                 FAX: +32(0)16 321979
Web:                                                   e-mail: patrick.vandijck@mmbio.vib-kuleuven.be
  http://www.meduniwien.ac.at/medbch/MolGen/k          Web: http://bio.kuleuven.be/mcb
uchler/
                                                       VANHEE Lies
TURNER Vincent                                         Ghent University
Institute of Microbiology                              Laboratory of Pharmaceutical Microbiology
University of Lausanne & University Hospital           Harelbekestraat 72
Center                                                 Ghent 9000 (BELGIUM)
Bugnon 48                                              Phone: +32 9 264 8142
Lausanne 1011 (SWITZERLAND)                            FAX: +32 9 264 8195
Phone: +41 21 314 40 62                                e-mail: Lies.Vanhee@UGent.be
FAX: +41 21 314 40 60
e-mail: vincent.turner@chuv.ch                         VAZQUEZ DE ALDANA Carlos
                                                       Instituto Microbiologia Bioquimica
URBAN Constantin Felix                                 CSIC
Molecular Biology                                      Campus Unamuno
Umeå University                                        Salamanca 37007 (SPAIN)
Sjukhusområdet 6 KL                                    Phone: +34 923 252092
Umeå 90187 (SWEDEN)                                    FAX: +34 923 224876
Phone: +46 (0)90 785 3341                              e-mail: cvazquez@usal.es
FAX: +46 (0)90 772 630
e-mail: constantin.urban@molbiol.umu.se                VERNAY Aurélia
Web: www.molbiol.umu.se                                Institute of Developmental Biology and Cancer
                                                       CNRS UMR6543
VANDENBOSCH Davy                                       Université Nice Sophia-Antipolis
Laboratory of Pharmaceutical Microbiology              Parc Valrose Cedex 02
Ghent University                                       Nice 06108 (France)
Harelbekestraat 72                                     Phone: +33 (0)4 92 07 64 64
Ghent 9000 (BELGIUM)                                   FAX: +33 (0)4 92 07 64 66
Phone: +32 (0)9 264 80 93                              e-mail: vernay@unice.fr
FAX: +32 (0)9 264 81 95                                Web:
e-mail: davy.vandenbosch@UGent.be                        http://www.unice.fr/isdbc/equipe/equipe.php?id=
                                                       12
VANDEPUTTE Patrick
Inst. of Microbiology                                  VOELZ Kerstin
Univ of Lausanne and Univ Hospital Center              School of Biosciences
Bugnon 48                                              University of Birmingham
Lausanne 1011 (SWITZERLAND)                            Edgbaston
Phone: +41 21 3144083                                  Birmingham B15 2TT (UNITED KINGDOM)
FAX: +41 21 3144060                                    Phone: +44 (0)121 41 45420
e-mail: patrick.vandeputte@etud.univ-angers.fr         FAX: +44 (0)121 41 45925
Web: http://www.chuv.ch/imul/                          e-mail: kxv468@bham.ac.uk
                                                       Web:
                                                         http://www.biosciences.bham.ac.uk/labs/may/Ho
                                                       me.html




                                                 208
WAGENER Jeanette                                      ZARAGOZA Oscar
Dermatology                                           Mycology Department
University Tübingen                                   National Center for Microbiology, ISCIII
Liebermeisterstrasse 25                               Crta. Majadahonda-Pozuelo, Km2
Tübingen 72076 (GERMANY)                              Majadahonda, Madrid 28220 (SPAIN)
Phone: +49 (0) 7071 29 86864                          Phone: + 34 91 822 3661
FAX: +49 (0) 7071 29 4405                             FAX: + 34 91 509 70 34
e-mail: jeanette.wagener@med.uni-tuebingen.de         e-mail: ozaragoza@isciii.es

WANG Yue                                              ZAVREL Martin
Genes and Development Division                        MBT
Institute of molecular and Cell Biology               Fraunhofer IGB
61 Biopolis Drive                                     Nobelstrasse 12
Singapore 138673 (SINGAPORE)                          Stuttgart 70569 (GERMANY)
Phone: +65 65869521                                   Phone: +49(0)711/970-4048
FAX: +65 67791117                                     FAX: +49(0)711/970-4200
e-mail: mcbwangy@imcb.a-star.edu.sg                   e-mail: zav@igb.fraunhofer.de
Web: http://www.imcb.a-star.edu.sg/php/wy.php
                                                      ZNAIDI Sadri
WEYLER Michael                                        Yeast Molecular Biology
Institut für Molekulare Infektionsbiologie            Institute for Research in Immunology and Cancer
Röntgenring 11                                        2950, chemin de Polytechnique
Würzburg 97070 (GERMANY)                              Montreal QC H3T 1J4 (CANADA)
Phone: +49 931 312127                                 Phone: +15143436111 ext. 0670
FAX: +49 931 312578                                   FAX: +15143437383
e-mail: Michael.Weyler@uni-wuerzburg.de               e-mail: sadri.znaidi@umontreal.ca
                                                      Web: www.iric.ca
WILSON Duncan
Microbial Pathogenicity Mechanisms
Hans Knoell Institute
Beutenbergstrasse, 11a
Jena 07745 (GERMANY)
Phone: +49(0)532 12 13
FAX: +49(0)3641 532 08 10
e-mail: Duncan.Wilson@hki-jena.de
Web: http://www.hki-jena.de/index.php

WOLKE Sandra
Molecular and Applied Microbiology
Hans Knoell Institute
Beutenbergstrasse 11a
Jena 07743 (GERMANY)
Phone: +49 (0)3641 532 1094
FAX: +49 (0)3461 532 0803
e-mail: Sandra.Wolke@hki-jena.de
Web: www.hki-jena.de

YADEV Nishant
Oral and Maxillofacial Medicine and Surgery
School of Clinical Dentistry, University of
Sheffield
19 Claremont Crescent
Sheffield S10 2TA (UNITED KINGDOM)
Phone: +44 (0) 114 271 7964
FAX: +44 (0) 114 271 7863
e-mail: n.yadev@shef.ac.uk




                                                209
                           INDEX OF AUTHORS

Abu Rayyan, W.: P70        Biswas, S.: P95              Coady, A.: P115
Abu-Abed, U.: P120         Bito, A.: P92                Coddeville, B.: P56
Alarco, A.: P66            Blatzer, M.: P29             Coelho, C.: P90
Albrecht, A.: P47          Blockhaus, C.: P118          Coenye, T.: P3, P38, P76,
Albuquerque de Andrade,    Bloem, K.: P104              P79
P.: P65, P128              Bonnin, A.: P107             Cole, G.: S14
Alcazar Fuoli, L.: P99     Boone, C.: S52               Coleman, D.: S12, P42,
Almeida, O.: P14           Boucher, G.: P66             P81
Alvarez, F.: P17           Bougnoux, M.: P96            Collins, M.: P137
Amarsaikhan, N.: P77       Bourgeois, C.: P111,         Connolly, L.: P37
Ambudkar, S.: P74          P119                         Cook, E.: S53
Anderson, J.: P141         Boyce, K.: S24               Correa-Bordes, J.: P34,
Andes, D.: S33             Brachhold, M.: P51           P35, P58
Andrews, B.: S52           Bragada, C.: P27             Correia, A.: P130
Andrianopoulos, A.: S24    Brakhage, A.: S41, P8,       Cossart, P.: S43
Arana, D.: P51             P110, P113                   Costa-de-Oliveira, S.:
Arkowitz, R.: P49, P50,    Breitenbach, M.: P92         P27, P93, P94
P55, P57, P59              Brinkmann, V.: P120          Coste, A.: P84, P98
Ashbaugh, A.: P137         Brown, A.: P41, P125         Cottier, F.: S22
Askew, C.: P138            Brul, S.: P100               Cowen, L.: P141
Avtandilyan, N.: P105      Brynda, J.: P82              Crittin, J.: P84
Azeredo, J.: P139          Busse, D.: S54               Cuenca-Estrella, M.: P123
Baddley, J.: P133          Butler, G.: P2, P5, P11,     Cunha, R.: P90
Bader, O.: P83, P96, P98   P21, P37                     Curado, F.: P90
Bakker, H.: P78            Byrnes III, E.: P6           Cushion, M.: P137
Balajee, S.: P133          Caballero-Lima, D.: P34,     Dalle, F.: P107
Barbosa, J.: P27           P35                          Dal-Rosso, R.: P128
Barker, K.: P66            Cabral, V.: P90              Davis, D.: P68
Bassilana, M.: P55, P57,   Cairns, T.: P106             Davis, M.: P17
P59                        Calabrese, D.: P83           de Boer, A.: P103
Bauer, R.: P118            Can, T.: P140                de Groot, P.: P100, P103
Bauerova, V.: P45          Cannon, R.: P87              de Haan, J.: P104
Bauser, C.: P83, P98       Canovas, D.: S24             De Koning, L.: P100
Beard, S.: S24             Cantero, P.: P9              De Koster, C.: P100
Bednarzcyk, D.: P59        Cao, F.: P33                 Deak, E.: P133
Begdullayev, A.: P71       Capella-Gutierrez, S.: P30   Decker, T.: P119
Ben-Zvi (Sharon), H.:      Caramalho, I.: P130          Deforce, D.: P79
P112                       Casadevall, A.: P65,         Dekkers, H.: P100
Bergmann, A.: P52          P123, P128                   Delepierre, M.: P56
Berkes, C.: P122           Cassone, A.: S34             Deneault, J.: S33
Berman, J.: P141           Chabrier-Rosello, Y.: P7     d'Enfert, C.: P28, P69,
Bertuzzi, M.: P40          Chakraborty, U.: P31         P96, P97
Bezerra, A.: P20, P63      Challacombe, S.: P134        Dierich, M.: P135
Bharucha, N.: P7           Chauvel, M.: P69             Dietl, J.: P116
Bignell, E.: S23, P40,     Chen, D.: P106               Ding, C.: P37
P52, P106                  Chen, J.: P32, P33           Diogo, D.: P69
Bildfell, R.: P6           Citiulo, F.: P42             Distel, B.: P104


                                      210
Dobson, C.: P97              Gonçalves, T.: P90          Hube, B.: P47, P101,
Dolejsi, E.: P45             Gonçalves-Rodrigues, A.:    P107, P109
Dörflinger, M.: P124         P27, P94                    Huerta-Cepas, J.: P30
Dostal, J.: P82              Gonzales Gonzales, M.:      Hughes, T.: S52
Doyle, S.: P15               P40                         Hung, C.: S14
Dulmage, K.: P141            Gonzalez-Novo, A.: P34,     Hussain, G.: S44
Dyer, P.: S13, P18           P35, P58                    Idnurm, A.: S25
Eaton, R.: P39               Gorantala, J.: P75          Isaac, D.: P115, P122
Edgerton, M.: S43            Gottar, M.: S44             Ischer, F.: P83
Einsele, H.: P118            Gow, N.: P125               Jackson, E.: P126
Ellison, C.: S14             Goyard, S.: P69             Jacobsen, I.: P101
Engblom, C.: P108            Gratz, N.: P64              Jain, C.: P121
Engel, J.: P78               Gross, U.: P98              Janbon, G.: P4, P13
Engström, Y.: P17            Grumaz, C.: P19, P54        Jansen, G.: P91
Epp, E.: P36, P91, P138      Grumbt, M.: P117            Jimenez, J.: P58
Ermert, D.: P120             Guida, A.: P5, P11, P37     Jöchl, C.: P15
Ernst, J.: P9, P10, P95      Guillas, I.: P57            Johnson, A.: S33
Fadda, G.: P83               Gunzer, M.: P110            Jones, L.: P62
Faz, F.: P104                Gutierrez-Escribano, P.:    Jorge, J.: P14
Fedorova, N.: S24, P106      P34, P35                    Jouault, T.: P136
Fernandez-Ruiz, E.: S43      Haas, H.: P29               Jungblut, P.: P120
Ferrandon, D.: S44           Hall, R.: S22, P39          Jungwirth, H.: P16
Ferrari, S.: P83, P85, P98   Hallet, M.: P91             Jürgensen, C.: P11
Fields, S.: P53              Hamari, Z.: P127            Kaffarnik, F.: P69
Filler, S.: S43              Hamed, S.: P95              Kavanagh, K.: P15
Findon, H.: P53              Harcus, D.: P91             Keeling, P.: S15
Fiori, A.: P43               Hardy, G.: P104             Keller, S.: P8
Firon, A.: P28, P69          Hartmann, T.: P52           Kelly, D.: P96
Follette, P.: P59            Hasenberg, M.: P110         Kelly, S.: P96
Fontaine, T.: P56            Hauser, N.: P98, P129       Keniya, M.: P87
Frank, S.: P6                Haynes, K.: S53, P53        Ketel, C.: P141
Frohner, I.: P16, P119       Heilmann, C.: P100          Klis, F.: P100
Fuller, H.: S13, P18         Heinekamp, T.: P8           Kniemeyer, O.: P77, P110
Gabaldon, T.: P16, P24,      Heitman, J.: S01, P6        Kornitzer, D.: P12, P49,
P30, P124                    Henriques, M.: P139         P50
Gacser, A.: P127             Hernandez, R.: P129         Kovarik, P.: P64
Galan-Diez, M.: S43          Hernday, A.: S33            Kraidlova, L.: P67
Galitski, T.: S51            Hiller, E.: P124            Krappmann, S.: P52
Gasparyan, A.: P105          Hnisz, D.: P44              Krauke, Y.: P46
Gastebois, A.: P56           Ho, H.: P53                 Krysan, D.: P7
Gehrke, A.: P110             Höfer, T.: S54              Kuchler, K.: P16, P44,
Gerardy-Schahn, R.: P78      Holcombe, L.: P126          P111, P119, P124, P135
Gfell, M.: P98               Holmes, A.: P87             Kuhns, M.: P96
Gibson, A.: P1               Homann, O.: S33             Kumar, A.: P7
Gildor, T.: P49, P50         Hood, M.: P1                Kurzai, O.: P118
Giraud, T.: P1               Hope, H.: P55               Küsel, J.: P54
Glaser, W.: P16, P44         Hruskova-                   La Fleur, M.: P80
Gobert, V.: S44              Heidingsfeldova, O.: P45,   Labrador, L.: P58
Goldman, W.: S42             P82                         Lagrou, K.: P3
Gomez-Raja, J.: P68                                      Lass-Flörl, C.: P135


                                       211
Latgé, J.P.: S32, P56      Mayer, F.: P109            Nelis, H.: P3, P38, P76,
Lavoie, H.: P138           McDonagh, A.: S53, P106    P79
Leach, M.: P41             McGee, C.: P21             Nesseir, A.: P69
Lee, A.: P91               McLachlan, A.: S24         Netea, M.: S45
Legrand, M.: P28           Meersseman, W.: P3         Nett, J.: S33
Leon, Z.: P36              Meireles, P.: P130         Nevitt, T.: P61
Leonhardt, I.: P47         Mellado, E.: P96, P98,     Nicholls, S.: P41
Lépine, G.: P36            P98, P99                   Nicola, A.: P128
Lermann, U.: P130          Melo, N.: P14              Niehus, S.: S44
Lesiak, I.: P135           Menshik, B.: P71           Nierman, W.: S24, P106
Lessing, F.: P110          Mezger, M.: P118           Niimi, M.: P87
Lettner, T.: P92           Mignon, B.: P117           Nikoyan, A.: P105
Lewis, K.: P80             Minuzzi, F.: P53           Nilsson, G.: P108
Lewit, Y.: P6              Miramon MartÌnez, P.:      Nobile, C.: S33
Li, W.: P6                 P47                        Nosanchuk, J.: P123,
Li, Y.: P33                Miranda, I.: P22           P127
Liégeois, S.: S44          Mitchell, A.: S33          Odds, F.: S11, P125
Lindemann, E.: P19, P54    Mitchell, T.: P6           Ofir, A.: P12
Linke, M.: P137            Mogensen, E.: P13          O'Gara, F.: P126
Lisboa, C.: P22            Monk, B.: P87              Ogel, Z.: P77, P140
Liu, H.: P32               Monod, M.: P117            O'Gorman, C.: S13, P18
Liu, T.: P66               Mooij, M.: P126            O'Keeffe, G.: P15
Ljungdahl, P.: P17         Moraes Nicola, A.: P65     Oliveira, R.: P139
Loeffler, J.: P118         Mora-montes, H.: P125      Osherov, N.: P112
Logue, M.: P2              Moran, G.: S12, P42, P81   Oshlack, A.: S24
Löhning, M.: S54           Moreno-Ruiz, E.: S43       Pablo-Hernando, M.: P58
L'Ollivier, C.: P107       Morillo-Pantoja, C.: P34   Padmanabhan, S.: P25
Lopez-Ribot, J.: P114      Morrissey, J.: P126        Pais, C.: P130
Lorenz, M.: P60            Morschhäuser, J.: S31,     Pantoja-Godoy, C.: P35
Lu, Y.: P32, P33           P48, P49, P50, P86, P130   Panwar, S.: P88
Luis, C.: P125             Moura, D.: P93             Paoletti, M.: S13
Lunderius Andersson, C.:   Mouyna, I.: P56            Parker, J.: P96
P108                       Moyes, D.: P134            Pase, L.: S24
Lynch, D.: P2              Moyrand, F.: P4, P13       Peck, S.: P69
Ma, B.: S53                Muhlschlegel, F.: S22,     Pedro, R.: P14
Macheleidt, J.: P8         P39                        Phan, Q.: S43
Mahanty, S.: P88           Mukhopadhyay, C.: P95      Pichova, I.: P45, P82
Mai, M.: P98               Mulhern-Haughey, S.: P5    Pierre, O.: P59
Majer, O.: P111, P119      Müller, M.: P119           Pina-Vaz, C.: P22, P27,
Manoharlal, R.: P74, P75   Mullick, A.: P36           P93, P94
Mao, X.: P32               Murciano, C.: P134         Pinto Silva, A.: P94
Marcet-Houben, M.: P24,    Murdoch, C.: P114          Pla, J.: P51
P30                        Nacken, W.: P120           Politz, S.: P121
Mariani, L.: S54           Naglik, J.: P134           Polonelli, L.: P81
Marr, K.: P6               Nailis, H.: P79            Posteraro, B.: P83
Martel, C.: P96            Nanagulyan, S.: P105       Poulain, D.: P136
Martinez-Esparza, M.:      Nantel, A.: S33, P72,      Prasad, R.: P73, P74, P75,
P136                       P138                       P95
Matskevitch, A.: S44       Nayyar, N.: S43            Prasad, T.: P95
May, R.: P102              Negri, M.: P139            Prusty Rao, R.: P121


                                     212
Puri, N.: P73               Schröppel, K.: P70         Strijbis, K.: P104
Puttnam, M.: S53            Schubert, S.: P86          Su, C.: P32, P33
Quintin, J.: S44            Schüll, T.: P48            Sudbery, P.: P62
Radbruch, A.: S54           Schüller, C.: P64          Sugareva, V.: P8
Ramirez, M.: P60            Schulz, E.: S54            Sullivan, D.: S12, P42,
Ramirez-Zavala, B.: P49,    Schwarzmüller, T.: P16,    P81
P50                         P44, P124, P135            Sun, J.: S43
Raymond, M.: P36, P66       Sehnal, M.: P70            Surprenant, J.: P91
Regenbogen, J.: P19         Seibold, M.: P96           Svidzinski, T.: P139
Rennemeier, C.: P116        Selander, C.: P108         Sychrova, H.: P46, P67
Rezacova, P.: P82           Sellam, A.: P72, P138      Synnott, J.: P5, P37
Ricardo, E.: P94            Selmecki, A.: P141         Taguchi, H.: P14
Rodrigues, A.: P22, P93     Selway, L.: P69            Talbot, N.: S02
Rodriguez-Tudela, J.:       Sen, M.: P26               Taylor, J.: S14
P123                        Setiadi, E.: P95           Thakur, J.: P25
Roemer, T.: S35             Shahana, S.: P125          Thiele, D.: P61
Roetzer, A.: P64            Sharma, M.: P74, P75       Thomas, D.: P91
Rogers, P.: P66, P86        Sharpton, T.: S14          Thornhill, M.: P114
Rohde, B.: P19, P83, P98    Shekhovtsova, E.: P131,    Tierney, L.: P111
Rossignol, T.: P69, P97     P132                       Tintelnot, K.: P96
Routier, F.: P78            Sheth, C.: P125            Tournu, H.: P67
Rowan, R.: P81              Shevchenko, M.: P131,      Turner, V.: P84, P89
Runglall, M.: P134          P132                       Urban, C.: P120
Rupp, S.: P19, P51, P54,    Shukla, S.: P74            Vagvolgyi, C.: P127
P124, P129                  Siddarthan, R.: P25        Valentine, B.: P6
Ryman, K.: P17              Sieglova, I.: P82          van den Burg, J.: P104
Sadhale, P.: P26            Sil, A.: P115, P122        Van Dijck, P.: P43, P67
Sai, S.: P21                Silva Dias, A.: P93        van Roermund, C.: P104
Sampaio, P.: P130           Silva, A.: P22, P27        van Vlies, N.: P104
Sanchez, M.: P58            Simenel, C.: P56           Vandenbosch, D.: P76
Sanglard, D.: P83, P84,     Simões, J.: P20, P63       Vanhee, L.: P3, P38
P85, P89, P96, P98, P99     Singh, A.: P70             Vauchelles, R.: P55
Sanguinetti, M.: P83        Singh, V.: P26             Vazquez de Aldana, C.:
Santos, M.: P20, P23, P63   Skibbe, M.: P101           P34, P35, P58
Sanyal, K.: P25, P31        Smyth, G.: S24             Veiga, E.: S43
Sapozhnikov, A.: P131,      Sohn, K.: P19, P54, P129   Vernay, A.: P57
P132                        Solis, N.: S43             Vignali, M.: P53
Sarazin, A.: P136           Sorgo, A.: P100            Vilanova, M.: P130
Sassi, N.: P107             Sosinska, G.: P100         Voelz, K.: P102
Saville, S.: P114           Soyler, A.: P140           Vogl, G.: P135
Schaller, M.: P103          Soyler, B.: P140           Wagener, J.: P103
Scheffold, A.: S54          Speth, C.: P135            Wanders, R.: P104
Scheynius, A.: P108         Stagljar, I.: P40          Wang, Y.: S21
Schindler, S.: P113         Staib, P.: P116, P117      Weber, J.: P117
Schmaler-Ripcke, J.: P8     Stajich, J.: S14           Weber, S.: P66
Schmalhorst, P.: P78        Stalder, D.: P55           Weig, M.: P96, P98, P103
Schmauch, C.: P49, P50      Steegborn, C.: S22, P39    Weindl, G.: P103
Schmid, M.: P120            Steele, C.: P133           West, L.: P53
Schrettl, M.: P29           Stevens, R.: P124          West, S.: P6
Schrieder, L.: S24          Stichternoth, C.: P10      Weyler, M.: P48


                                      213
Whiston, E.: S14
Whiteway, M.: P36, P91,
P138
Wilson, D.: P109
Wolfe, K.: P2
Wolke, S.: P110
Würzner, R.: P135
Xie, C.: P80
Yadev, N.: P114
Yun, M.: P121
Zaragoza, O.: P123
Zaugg, C.: P117
Zavrel, M.: P129
Zeidler, U.: P92
Zhu, W.: S43
Zipfel, P.: P113
Znaidi, S.: P66
Zychlinsky, A.: P120




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