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 evaluating the genetic diversification of Candida albicans and how it contributes to the virulence of this species. Furthermore, we are deciphering the molecular mechanisms that are responsible for the formation of biofilms by Candida albicans (adhesion, morphogenesis, extracellular matrix production) and the original physiology of these three-dimensional microbial structures (antifungal resistance). To this aim, we use both genomic and molecular genetic approaches. Furthermore, we have pursued a detailed study of a critical process in the life cycle of filamentous fungi, namely spore germination, and are conducting an exhaustive search of essential genes in the fungal pathogen of humans, Aspergillus fumigatus.
Candida albicans: genomics, biofilms and epidemiology (M.-E. Bougnoux, M. Chauvel, D. Diogo, A. Firon, S. Goyard, E. Moreno-Ruiz, G. Ortu and 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 and re-annotation of the C. albicans genome; the analysis of the genomes of Aspergilli (A. nidulans, A. fumigatus, A. oryzae and more recently A. niger) in the framework of international consortia.
Furthermore, our group is coordinating the Marie Curie Research and Training Network Galar Fungail 2 that studies the interaction of Candida albicans with the host using genomic, molecular and cellular approaches.
a. Biofilm formation in Candida albicans [M. Chauvel, A. Firon, S. Goyard, E. Moreno-Ruiz and 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. We have focused on the inactivation of 48 over-expressed genes and phenotypic characterization of the resulting mutant strains has been accomplished in 2005. Our results show that the yeast-to-hypha transition is critical for biofilm formation by C. albicans. However, 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 genes that are necessary for efficient biofilm formation will be pursued.
G. janbon et I. Iraqui (Molecular Mycology Unit) 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 adhesins necessary for biofilm formation. We have investigated the role of Yak1 in C. albicans and shown that yak1 is necessary for morphogenesis and consequently biofilm formation by C. albicans. We are pursuing the characterization of Yak1 in the framework of a Transversal Research Program involving the Molecular Mycology Unit, the Organic Chemistry Unit, the Structural Biochemistry Unit and the Production of antibodies and recombinant proteins platform. We are also attempting the identification of Yak1 targets through proteomics in collaboration with the Proteomics platform.
d. Molecular epidemiology of C. albicans [M.-E. Bougnoux, D. Diogo and 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 or seven 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, London).
MLST has been applied to several collections of clinical and commensal C. albicans strains through a collaboration with the Sequencing platform of Pasteur Génopole® Ile-de-France. Our results indicate that loss-of-heterozygosity events are frequent and responsible for microevolution of diploid C. albicans within clonal groups. Our results also suggest that mating of C. albicans that was recently demonstrated in the laboratory could also occur in Nature. The occurrence of a high level of heterozygosity in natural populations of C. albicans suggests that loss-of-heterozygosity events are counter-selected in the environment and we aim to investigate this hypothesis. Interestingly, our analysis of commensal isolates of C; albicans has enabled the identification of closely related strains that differ through micro-evolutions in a single individual or in individuals within a family, indicating rapid evolution of C. albicans within the gastro-)intestinal tract. The nature and extent of these micro-evolutions is currently investigated.
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 have characterized the signaling pathways mediating the recognition of environmental signals triggering germination and their transduction towards the resumption of various metabolic processes that are necessary for germination.
Recent work has focused on the characterization of the mechanisms regulating the activity of adenylate cyclase and cAMP-dependent protein kinase at the onset of spore germination. We have conducted an in silico analysis of the A. nidulans, A. fumigatus and A. niger genomes in order to identify components of G-protein and cAMP-mediated signaling. This study has shown a striking diversity of the putative G protein-couple receptors (GPCRs, 13-16 according to the species). A similar observation is made in other filamentous ascomycetes in contrast to yeasts that have only a limited number of GPCRs. Other signaling components acting downstream of GPCRs are highly conserved in structure and numbers. These observations probably reflect the abilities of filamentous ascomycetes to adapt to various ecological niches and to integrate diverse environmental cues into highly conserved cellular processes. Additionally, 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 activates 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). Finally, we have completed the characterization of the regulatory subunit of the cAMP-dependent protein kinase - PkaR - and shown that it regulates various aspects of A. nidulans development including germination, hyphal growth and conidiogenesis.
Systematic identification of essential genes in the fungal pathogen of humans, Aspergillus fumigatus (M. Lecomte, L. Simon and 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 is now partially automated and is used to systematically identify A. fumigatus essential genes.
Photo 1 : Top-view of a biofilm formed by Candida albicans on a plastic surface. Hyphal cells are embedded in exopolymeric matrix material (Scanning Electron Microscopy; collaboration Adeline Mallet et Marie-Christine Prévost, Plate-forme de Microscopie électronique)
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