Functional Characterisation of Candida Glabrata Open Reading Frames with no Orthologue in Saccharomyces Cerevisiae
Thesis or dissertation
University of Exeter
Candida glabrata is a significant and increasingly common pathogen of humans yet its mechanism of virulence remains unclear. Comparative genomic studies revealed that C. glabrata is more closely related to the non-pathogenic yeast Saccharomyces cerevisiae and that both these genomes are distinct from C. albicans. In order to explore C. glabrata virulence attributes, C. glabrata ORFs with no orthologue in S. cerevisiae were studied since these ORFs may have accompanied the adaptation of C. glabrata to the human host. Reciprocal best hit searches identified C. glabrata ORFs with no S. cerevisiae orthologue. A barcoded deletion library targeting 65 C. glabrata-specific ORFs was constructed. To functionally characterise the deletion library, mutants were tested for fitness and phenotypically screened to identify gene products required for growth in response to biologically relevant stresses. As such, novel phenotypes associated with the deletion of previously uncharacterised ORFs were uncovered. Mutants were also tested for infection-related properties including biofilm formation, antifungal agent susceptibility and for virulence in a Drosophila melanogaster infection model, resulting in the identification of two C. glabrata-specific ORFs, CAGL0K05687g and CAGL0H01749g, which were required for virulence. Three ORFs with notable phenotypes were taken forward for further characterisation. An adapted genome-wide synthetic genetic interaction approach was used to create genetic interaction networks for C. glabrata ORFs over-expressed in S. cerevisiae. Genetic interaction analysis of a C. glabrata chromatin remodeler CAGL0D05434g revealed a role for this ORF in metal ion homeostasis and DNA damage repair. Genetic interaction profiling for an oxidoreductase encoded by CAGL0K05687g was used to reveal mechanisms related to transport by which this ORF may be required for virulence.
PhD in Biological Science