Unit: Fungal Biology and Pathogenicity - INRA USC 2019

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, M. Chauvel, S. Goyard, P. Knechtle, M. Ornatowska, G. Ortu 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.

We are also involved in the in silico analysis of fungal genomes: the maintenance and curation of the C. albicans genome database CandidaDB http://genolist.pasteur.fr/CandidaDB/ , the re-annotation of the C. albicans genome and the analysis the genomes of Aspergilli (A. nidulans, A. fumigatus and A. oryzae) both in the framework of international consortia.

Furthermore, our group is coordinating the Marie Curie Research and Training Network Galar Fungail 2 ;http://www.galarfungail.org that studies the interaction of Candida albicans with the host using genomic, molecular and cellular approaches.

a. Formation de biofilms par Candida albicans [S. Aubert, M. Chauvel, S. Goyard, P. Knechtle, M. Ornatowska et G. Ortu]

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 directly 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.

Comparative transcript profiling of planktonic cultures and biofilms of C. albicans has resulted in the identification of a large number of genes that are over-expressed in biofilms. We are currently evaluating the contribution of these genes through the construction and analysis of C. albicans strains with corresponding null mutations. 48 genes have been inactivated in 2004. Characterization of the resulting strains shows that the yeast-to-hypha transition is critical for biofilm formation by C. albicans. Furthermore, several mutant strains produce biofilms with a reduced cohesiveness, suggesting that the corresponding genes might be involved in the production of the biofilm extracellular matrix. The detailed characterization of those genes that are necessary for efficient biofilm formation will be pursued.

G. janbon et I. Iraqui (Molecular Mycology Unit http://www.pasteur.fr/recherche/RAR/RAR2003/Mymol.html ) have established a role for the Yak1 protein kinase in biofilm formation by C. glabrata. Yak1 appears to regulate telomeric silencing and consequently the expression of genes encoding adhesions that are necessary for biofilm formation. We have investigated the role of Yak1 in C. albicans and preliminary results show that it is also necessary for biofilm formation.

d. Molecular epidemiology of C. albicans [M.-E. Bougnoux, D. Diogo 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. MLST is highly portable and data can be exchanged through a database for C. albicans MLST data (http://calbican.mlst.net) that we have established and maintain 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 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 have focused 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 in response to a carbon source. GanB has been shown to activate cAMP synthesis at the very onset of germination. This function of GanB requires the SfaD/Gβ et GpgA/Gγ subunits and is modulated by the RGS protein RgsA (Collaboration J. Yu, U. Wisconsin, USA).

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 may hence serve as antifungal targets. 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. This approach has been partly automated allowing the systematic identification of A. fumigatus essential genes. Current research is also aimed at establishing a tetracycline-regulatable promoter for conditional expression of A. fumigatus essential genes..

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

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

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