Unit: Fungal Biology and Pathogenicity - INRA

Director: d’Enfert, Christophe

We study several biological processes in fungi of the genus Candida and Aspergillus with the aim of developing new strategies for the control of fungal growth. We are in particular interested in deciphering the molecular mechanisms that are responsible for the formation of biofilms by Candida albicans. To this aim, we use both genomic and molecular genetic approaches. Furthermore, we are pursuing a detailed study of a critical process in the life cycle of filamentous fungi, namely spore germination, and conducting an exhaustive search of essential genes in the fungal pathogen of humans, Aspergillus fumigatus.

Candida albicans: genomics, biofilms and epidemiology (S. Aubert, M.-E. Bougnoux, F. Cottier, S. Garcia-Sanchez, S. Goyard, P. Knechtle et C. d'Enfert)

Candida albicans is currently the main fungal pathogen of humans. In particular, C. albicans is responsible for systemic infections in patients, which are severely immuno-compromized and receive broad-spectrum antibiotherapy. Systemic candidiasis are associated with a high mortality despite the availability of antifungal treatments. A better management of C. albicans infections will require an understanding of their epidemiology and physio-pathology as well as the development of new drugs. Our research is therefore interested in the epidemiology of C. albicans infections and in the study of processes related to pathogenicity through genomic approaches.

a. Genomic tools for the study of Candida albicans [S. Garcia-Sanchez, S. Goyard, P. Knechtle et C. d'Enfert]

C. albicans is an obligate diploid. Consequently, its study through reverse genetics is relatively tedious. Since a few years, a draft of the C. albicans genome has been made available to the scientific community by the Stanford Genome Technology Center. We taken advantage of this information to develop genomic tools that allow to address several aspects of the biology of C. albicans using large-scale approaches (transcript profiling, proteomics) : a genomic database CandidaDB http://genolist.pasteur.fr/CandidaDB/, and DNA-arrays that allow a simultaneous analysis of the transcriptional status of almost all C. albicans genes. We are currently involved in an international re-annotation phase of the latest version of the C. albicans genome made available by the Stanford Genome Technology Center in May 2002. This work will allow the release of an update of CandidaDB in january 2004.

Comparison of the C. albicans genome to those of other ascomycetous pathogens or saprophytes has resulted in the identification of a subset of genes that seem typical of pathogenic and/or hyphal ascomycetes and could play a critical role in the biology of these fungi (Collaboration P. Philipsen, U. Bâle). Three such genes, two encoding phospholipases and one a nucleotide exchange factor, are currently being characterized.

Our group is also involved in several collaborations for transcript profiling of C. albicans strains (J.. Pla, U. Madrid ; A. Mitchell, Columbia U., New York ; J. Perez-Martin, U. Madrid).

b. Formation de biofilms par Candida albicans [S. Aubert, F. Cottier, S. Garcia-Sanchez, S. Goyard et F. Mateen]

Biofilms are microbial communities that develop in association with a surface (Photo 1). They form a protected environment where microorganisms adopt a specific physiology. In the case of pathogenic yeasts, biofilm formation has been observed on different medical devices (prosthesis, catheters) and is associated to several forms of disease (e.g. stomatitis). Inside the biofilm, Candida cells have an elevated resistance to antifungals and this often results in sequels following an apparently successful antifungal treatment. Therefore, a better understanding of the molecular mechanisms underlying bofilm formation is necessary in order to develop novel strategies for the detection or prevention of biofilms. and achieve a better management of Candida infections.

Since 2001, we have developed a study of biofilm formation by Candida sp. in the framework of a Programme Transversal de Recherche with the Unité de Mycologie Moléculaire http://www.pasteur.fr/recherche/RAR/RAR2003/Mymol.html and the Groupe de Génétique des Biofilms http://www.pasteur.fr/recherche/unites/Ggb/ . Our objectives are to decipher the molecular mechanisms involved in biofilm formation, to understand the physiology of biofilm cells and to propose novel strategies for the detection and prevention of biofilms. In this context, our group has established several models of biofilm formation for C. albicans and Dans cette optique, notre groupe a mis en place différents modèles de biofilm pour Candida albicans et initiated the identification of genes specifically expressed in mature biofilms. This has been performed through transcript profiling of biofilm and planktonic cultures grown under a variety of physiological conditions and using strains of C. albicans differing by their ability to achieve the yeast-to-hypha transition. The main conclusion of this study is that biofilm formation represents a specific developmental event associated with the xpresion of a specific subset of genes. Interestingly, it appears that the transcriptome of biofilms is in a particular state that is only modestly sensitive to variations in the environment.

This approach has enabled the identification of a large group of genes which are over-expressed in biofilms with respect to planktonic cultures. Our current research is aimed at evaluating the contribution of these genes to biofilm formation and physiology through the construction of C. albicans strains with null mutation in one of these genes. In particular, we have focused on two genes, PGA59 and PGA62, which encode candidate glycosyl phosphatidylinositol (GPI)-anchored proteins. The cell surface localization of the two corresponding proteins has been confirmed (Photo 2). C. albicans cells with a deletion of the two PGA62 alleles show a defect in biofilm formation which might result from a defect in the yeast-to-hypha transition.

d. Molecular epidemiology of C. albicans [M.-E. Bougnoux et C. d'Enfert]

We have established Multi Locus Sequence Typing (MLST) for C. albicans and shown that it is highly reproducible and discriminatory. The MLST scheme is based on the sequencing of six loci which show intra-specific variations. In collaboration with the group of F. Odds (U. Aberdeen, Ecosse) which has established a similar typing scheme, we have re-analyzed 90 C. albicans strains using data for 10 loci and proposed a consensus typing scheme which is based on the sequencing of seven loci and show a higher discriminatory power than the two previously published schemes. In addition, we have established a database for C. albicans MLST data (http://calbican.mlst.net) in collaboration with the group of B. Spratt (Imperial College, Londres).

MLST has been applied to several collections of clinical and commensal C. albicans strains through a collaboration with the Sequencing facility of the Génopole Institut Pasteur http://www.pasteur.fr/recherche/RAR/RAR2003/Ptgen.html>. This study indicates that MLST compares well with the Ca3 fingerprinting method commonly used for typing C. albicans strains. 5 major groups of strains similar to those defined through Ca3 typing have been identified. Remarkably, some recombined genotypes have been observed suggesting that mating of C. albicans that was recently demonstrated in the laboratory could also occur in Nature. From an epidemiological standpoint, the study of isolates from clinical, commensal or animal origin suggests that strains associated to invasive infections are not genetically clustered. Moreover, typing of strains obtained from several Intensive Care Units in France idemonstrates not only intra-hospital strain transmission but also inter-hospital transmission.

Aspergillus nidulans spore germination (A. Lafon, M.K. Chaveroche and C. d'Enfert)

Filamentous fungi of the genus Aspergillus have been widely used for biotechnological processes but are also responsible for human disease. Spore germination is a key developmental step in the fungal life cycle and is critical for the establishment of the fungus in a new environmental niche. Our aim is to define the molecular and biochemical events that are involved during the early stages of spore germination. In particular, we wish to characterize the signaling pathways mediating the recognition of environmental signals necessary for germination and their transduction towards the resumption of various metabolic processes that are necessary for germination.

Our work has resulted in the demonstration that cAMP signaling is involved in the early stages of spore germination in the model filamentous fungus Aspergillus nidulans. Present research is aimed at identifying the mechanisms of activation of adenylate cyclase and the targets of cAMP during spore germination. In this respect, we are focusing our research on heterotrimeric G-proteins and G-protein coupled receptors. We have shown that only one of the three A. nidulans Gα subunits, namely GanB, is important for the control of spore germination. Furthermore, we have identified Gβ et Gγ subunits and GTP-exchange factors involved in the control of spore germination (Collaboration J. Yu, U. Wisconsin, USA).

Analysis of a collection of A. nidulans ESTs (Expressed Sequence Tags) produced by the Genomics Platform (PT1) of the Institut Pasteur Génopole http://www.pasteur.fr/recherche/RAR/RAR2003/Ptgen.html has been completed and will enable the development of DNA-arrays which will be used to study in greater details A. nidulans strains with defect in cAMP signaling.

Systematic identification of essential genes in the fungal pathogen of humans, Aspergillus fumigatus (L. Simon et C. d'Enfert)

Aspergillus fumigatus is the causative agent of invasive aspergillosis, which represents the second cause of death resulting from fungal infections in hospitals. Invasive aspergillosis is associated with a high mortality. This is mostly due to the poor sensitivity of the currently available diagnostic tests and the sole use of amphotericin B therapy, which has deleterious side effects. Analysis of candidate virulence factors by reverse genetics has not been successful for the understanding of the pathogenic processes. We have therefore focused our interest towards the development of novel molecular tools that could be used to identify through insertional mutagenesis genes that are essential for growth of A. fumigatus under laboratory conditions and/or during invasion of the host and may hence serve as antifungal targets. This program has been performed in collaboration with Bayer CropScience http://www.bayercropscience.com>.

We have established a novel technology for the identification of essential genes in A. fumigatus that combines transposon mutagenesis of an artificial diploid with haploidization through parasexual genetics. In a preliminary screen, we have identified 29 loci which are essential to A. fumigatus growth. Current research is aimed at up-scaling this technology in order to develop a compendium of A. fumigatus essential genes.

Photos :

Photo 1 : Biofilm of Candida albicans formed on a plastic surface (Scanning Electron Microscopy; collaboration E. Arbeille, Université de Tours)

Photo 2 : Cell surface localization of a Pga59-GFP hybrid protein (Confocal microscopy, Cente d'Imagerie Dynamique, Institut Pasteur)

Keywords: Aspergillus nidulans, Aspergillus fumigatus, Candida albicans, cAMP, kinase, signal transduction, germination, spore, conidia, antifungal, target, transposon, DNA chip, DNA array, biofilm, epidemiology, Multi-Locus Sequence Typing, genomics, transcriptome, transcript profiling"

Activity Reports 2003 - Institut Pasteur

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