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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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com 2 Smurfit Institute of Genetics, University of Dublin, Trinity College, Dublin 2, Ireland. e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org, 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: email@example.com 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 . 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.  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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.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: firstname.lastname@example.org 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 μg/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: email@example.com 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org, 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 . 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  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 ), as well as >30 predicted hyperactive or dominant- negative alleles of these genes from a tetracycline-inducible promoter  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- email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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%), С.guilliermondii (2%), C.famata (2%), C.zeylanoides (2%), C.rugosa (2%), С.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: email@example.com 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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org, 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 . 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 . 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com,firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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- email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org, 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-γ, 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: email@example.com 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: firstname.lastname@example.org, 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org 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: email@example.com, 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: firstname.lastname@example.org, 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: email@example.com, 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org Phone: +33 (0)4 92 076425 Web: http://www3.imperial.ac.uk FAX: +33 (0)4 92 076466 e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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 :firstname.lastname@example.org Phone: +77051900585 FAX: +77272206467 ANDREWS Brenda e-mail: email@example.com 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: firstname.lastname@example.org Nice 06108 (France) Web: http://www.utoronto.ca/andrewslab/ Phone: +33 (0)4 9207 6464 FAX: +33 (0)4 9207 6466 e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org FAX: +43 (0)662 8044 144 e-mail: email@example.com 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: firstname.lastname@example.org Phone: +44 1224 555928 Web: http://www.cbs.umn.edu/labs/berman/ FAX: +44 1224 555844 e-mail: email@example.com 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: firstname.lastname@example.org FAX: +33 (0)1 45 68 89 38 e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com Phone: 31205256970 Web: www.iss.it FAX: 31205256971 e-mail: firstname.lastname@example.org 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: email@example.com FAX: +353-1-2837211 e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com FAX: +41 (0)21 314 40 60 e-mail: firstname.lastname@example.org 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: email@example.com Phone: +351 239 857772 FAX: +351 239 822776 COENYE Tom e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org E-mail: email@example.com 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: firstname.lastname@example.org FAX: +1 919 962 8103 e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com Jena 07745 (GERMANY) Web: www.intercell.com Phone: +49 3641 532 1247 FAX: +49 3641 532 0809 e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com Phone: +49 (0)711 970 4171 FAX: +49 (0)711 970 4200 HALL Rebecca e-mail: firstname.lastname@example.org 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: email@example.com FAX: 004962215451487 Web: http://www.kent.ac.uk/bio/muhlschlegel/ e-mail: firstname.lastname@example.org 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: email@example.com FAX: +353 (0)21 4903101 e-mail: firstname.lastname@example.org 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: email@example.com Phone: +420 220 183 249 FAX: +420 224 310 090 HEINEKAMP Thorsten e-mail: firstname.lastname@example.org 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: email@example.com London SW7 2AZ (UNITED KINGDOM) Web: http://www.hki-jena.de Phone: +44 02075947409 FAX: +44 02075943095 e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org Phone: +972 (0)4 829 5258 FAX: +972 (0)4 829 5254 e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org FAX: +497119704200 e-mail: email@example.com 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: firstname.lastname@example.org FAX: +43 (0) 4277 9618 e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com FAX: 412-268-7129 Web: www.mfpl.ac.at e-mail: firstname.lastname@example.org 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: email@example.com Phone: +33 1 45 68 83 56 FAX: +33 1 45 68 84 20 MARCET-HOUBEN Marina e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org Immunology Institute of Life Science, Swansea University Singleton Park Swansea SA2 8PP (UNITED KINGDOM) Phone: +44 01792 368578 FAX: +44 01792 301022 e-mail: email@example.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: firstname.lastname@example.org- e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com Phone: +44 (0)1224 555927 FAX: +44 (0)1224 555844 ODDS Frank e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.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: firstname.lastname@example.org Science Centre (West) Belfield, Dublin 4 (IRELAND) Phone: +353 (0)1 716 2350 FAX: +353 (0)1 716 1153 e-mail: email@example.com 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: firstname.lastname@example.org FAX: + 33 (0) 3 88 60 69 22 e-mail: email@example.com 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 : firstname.lastname@example.org FAX: +0049 931 312578 e-mail: email@example.com 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 : firstname.lastname@example.org e-mail: email@example.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: firstname.lastname@example.org e-mail: email@example.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: firstname.lastname@example.org FAX: 001-508-831-5936 Web: http://www.pasteur.fr/bpf e-mail: email@example.com 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: firstname.lastname@example.org FAX: + 91-11-26741081 e-mail: email@example.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: firstname.lastname@example.org 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: email@example.com E-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org FAX: +49 (0)7071 29 5440 Web: http://www.ua.pt/ii/rnomics/ e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com Phone: +44(0)1224 555878 Web: www.fmup.pt FAX: +44(0)1224 555844 e-mail: firstname.lastname@example.org 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: email@example.com FAX: + 91-11-26741081 Web: http://www.ua.pt/ii/rnomics e-mail: firstname.lastname@example.org 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: email@example.com FAX: +7 495 330 40 11 Web: http://www.ucd.ie/biochem/gb/Lab/ e-mail: firstname.lastname@example.org 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: email@example.com FAX: +74953304011 e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com FAX: + 510 642 4995 Web: http://www.sibs.ac.cn/ e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org 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: email@example.com FAX: +46 (0)90 772 630 e-mail: firstname.lastname@example.org 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: email@example.com 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: firstname.lastname@example.org FAX: +44 (0)121 41 45925 Web: http://www.chuv.ch/imul/ e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org e-mail: email@example.com 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: firstname.lastname@example.org 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: email@example.com 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 214 215
"Human Fungal Pathogens May 2-8_ "