Comprehensive gene deletion study to identify cell wall organisation and structure in Candida glabrata

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 inhouse 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