Abstract:
The pathogenic opportunistic fungus Candida albicans is a leading causative agent of death in immunocompromised individuals. The fungus has a wide arsenal of cell surface and secreted virulence factors that contribute to its pathogenesis. There is a growing trend of drug resistance to various antifungal agents such as fluconazole which belongs to the azoles family, and caspofungin, an echinocandin. The purpose of this study is to induce resistance to such drugs in a sensitive C. albicans reference strain ATCC5215 by culturing in increasing drug concentrations followed by whole genome sequencing to determine molecular mechanisms of resistance. In addition, strains will be assayed for pathogenicity attributes such as ergosterol and chitin content, growth rate, virulence, and biofilm-forming capabilities. We observed a sequential increase in ergosterol and chitin content in fluconazole resistance-induced isolates by 78% and 44% respectively in the most resistant isolate compared to the control. Such an increase thickens the cell surface and prevents drug entry. Developing resistance to antifungal drugs can impose a fitness trade-off on the Candida albicans cells, whereby resistance mechanisms, such as efflux pumps or mutations in drug targets, lead to reduced growth rates, biofilm formation and reduced virulence which is what we observed in our study. Biofilm formation decreased by 21.76% in our most resistant isolate. Furthermore, whole genome sequencing data revealed mutations in genes involved in resistance and pathogenicity attributes such as ERG11, CHS3, GSC2, CDR2, CRZ2, and MSH2. Some of these mutations were previously documented and many are novel undocumented mutations in key genes. An interesting aspect of our study is the increase in the number of mutations in key genes with a sequential increase of drug selective pressures as the organism tries to increase its odds of adapting to an inhospitable environment. In the ALS4 gene for example, we observed 2 mutations in the susceptible strain and 5 mutations in the resistant strain. This is the first study of its kind to induce drug resistance followed by a genotypic and phenotypic analysis of the isolates, with the aim of determining mechanisms of resistance to antifungal drugs.
