Abstract

Candida albicans, an opportunistic pathogenic fungus, is a leading cause of nosocomial infections. In last two decades, the rise of antifungal resistance calls for better understanding of genetic networks responsible for mechanisms of resistance. Genetic analyses have been fundamental in unveiling cell adaptation mechanisms and transcriptional rewiring. High-throughput data from the Gene Replacement and Conditional Expression 1.0 collection of gene inactivations identified strains resistant to the common antifungal fluconazole, including RAP1, ERG3 and HCS1. Using CRISPR-Cas9, we generated targeted single and double deletion mutants of these genes and studied their response to fluconazole treatment. Our study reveals functional diversity in strains lacking RAP1. RAP1 deficiency also causes colony size heterogeneity and morphological changes to pseudohyphae. We also utilized an activated transcription factor library to perform a high-throughput screenings with different stressors including pH, heavy metal tolerance, and fluconazole. We were able to find interesting phenotypic responses such as four TFs whose activation conferred resistance to hydroxyurea, eight involved in pH response and six in temperature response. Moreover, one strain with transcription factor Adr1 activated showed multidrug resistance, and we identified a change in function rewiring from control of ergosterol biosynthesis in Candida albicans to control of alcohol and fatty acid metabolism in Saccharmocyes cerevisiae. Our findings thus highlight two genes with involvement in fluconazole response which can provide new insight to the transcriptional regulation of C. albicans and potentially direct more effective treatments.