Determinants of Fluconazole Resistance Evolvability in Candida auris

About This Grant

PROJECT SUMMARY/ABSTRACT The expansion of antimicrobial resistance has been called "one of the greatest threats we face as a global community" by the United Nations. Efforts to combat antimicrobial resistance tend to target the mechanisms of resistance, while less is understood regarding the biological basis for the evolution of these mechanisms. This discrepancy becomes apparent in examining pathogens such as Candida auris, an emerging "superbug" heralded by health agencies for its high rates of drug resistance. Resistance in C auris is entirely driven by acquired resistance mutations, but these mutations occur substantially more frequently in C auris than in other pathogenic Candida species. This suggests an unexplored biological mechanism for the acquisition and maintenance of resistance mutations in C. auris. To interrogate this mechanism, in Aim 1, I will investigate the evolutionary patterns that lead to accumulation of resistance mutations in a susceptible population. I will compare resistance evolvability in susceptible clinical isolates against resistant isolates genetically reverted to a susceptible or immediately "pre-resistant" state. These experiments will delineate the scope of evolvability in precipitating C auris antifungal resistance. To explore the extent to which these mechanisms contribute to the emergence of specific localized resistance mutations in an evolving population, I will develop and employ a high-resolution single-cell transcriptomics approach to track the emergence of resistant subpopulations and compare evolutionary patterns between lineages more likely to evolve resistance with those less likely. In Aim 2, I will investigate the physiological compensation required to tolerate and maintain resistance mutations despite expected perturbations to core biological processes associated with such mutations. Here I will compare the impact of resistance mutations in regulatory pathways to isolated changes in the effectors of these pathways. To this end, I will develop and employ a genome-scale CRISPR disruption library to identify genetic processes conditionally essential under acquired resistance. This will identify biological processes that recognize and respond to resistance mutations, and I will then evaluate their regulation in naturally resistant populations to implicate their essentiality for the maintenance of resistance in nature. These studies will explain how an emerging multidrug-resistant pathogen of substantial public health concern has evolved antimicrobial resistance on a scale order of magnitude higher than its related species, potentially exposing fundamental evolutionary biology that will inform efforts to combat resistance. Furthermore, this research will provide a conduit for me to take advantage of expertise at St Jude to build mentored experience in cutting edge genetic techniques such as custom library design, single-cell RNA seq, saturating CRISPR screening, and associated analyses. Developing these techniques for C. auris will represent their most expansive application in fungal pathogens to date and will provide a comprehensive toolset to prepare me for an innovative independent research career.