Genomic analysis of antifungal drug resistance and tolerance, and centromere structure in budding yeasts

Candida parapsilosis is an emerging opportunistic fungal pathogen increasingly associated with fluconazole treatment failures. In Chapter 2 of this thesis, we examined both fluconazole resistance and tolerance in 386 C. parapsilosis isolates. Using disk diffusion assays we measured the prevalence of resistance and tolerance in C. parapsilosis. Findings revealed that 25.9% of isolates exhibited high fluconazole tolerance. Whole-genome sequencing showed that many tolerant isolates belonged to a single phylogenetic clade, suggesting that tolerance is genetically determined. Temperature and nutrient stress influenced tolerance in a strain-specific manner. Inhibition of the calcineurin pathway and Hsp90 significantly reduced tolerance which suggests these stress response pathways play a role in C. parapsilosis tolerance. In Chapter 3, we investigated the interaction between fluconazole and fluphenazine, a phenothiazine antipsychotic drug. Similar to C. albicans, fluphenazine antagonised fluconazole activity in C. parapsilosis in a concentration- and strain-dependent manner. CRISPR-Cas9 editing revealed that this antagonism was mediated by Cdr efflux pumps regulated by the transcription factor Tac1. Simultaneous disruption of multiple efflux pump genes was required to significantly reduce antagonism. In some highly tolerant strains, fluphenazine displayed a synergistic effect at higher fluconazole concentrations. In Chapter 4, we analysed centromere evolution across budding yeasts in the Saccharomycodales and Phaffomycetales. Using Hi-C mapping and ChIP-seq, we observed that centromere structures varied among yeasts and included sequence-defined, inverted repeat, and Ty element-associated centromeres. However, we show that centromere locations remained largely conserved. Our results suggest that point centromeres arose earlier than previously recognised, in a common ancestor of Saccharomycetales, Saccharomycodales, and Phaffomycetales. Overall, the work in this thesis gives us better insights into antifungal drug responses and centromere evolution in budding yeasts. These findings have both clinical and evolutionary significance.