Project Summary Candida auris has recently emerged as a cause of invasive infections worldwide that are associated with high mortality rates despite treatment with echinocandins, the frontline class of antifungal agents, and other drugs. C. auris poses unique challenges due to its propensity for antifungal resistance, and ability to persist in hospital environments and cause long-tern outbreaks. C. auris are haploid yeasts that are evolutionarily divergent from diploid Candida like C. albicans and most pathogenic spp. Strains fall in 5 phylogeographic clades. Most infections are caused by clade I, III or IV strains. There is mounting evidence for genetic diversity within clades, including core genome single nucleotide polymorphisms, karyotype alterations, gene copy number variations, polyploidy and aneuploidy. Strains within a given clade also demonstrate differences in antifungal susceptibility, virulence attributes and pathogenicity. To date, few specific genes have been validated as virulence determinants, and pathogenesis and echinocandin tolerance remain poorly understood. Mechanisms of tolerance, resistance, virulence and pathogenesis cannot necessarily be extrapolated from other Candia spp., or between C. auris clades. The long-standing paradigm is that almost all Candida infections of blood or other normally sterile sites stem from a single, clonal organism. However, in preliminary studies, we have shown that at least some C. auris and C. glabrata strains recovered from positive blood cultures from individual patients at our center exhibit unrecognized genotypic and phenotypic diversity. Our data suggest a new, population-based paradigm for Candida infections. The objectives of this project are to characterize the genetic and phenotypic diversity of C. auris strains recovered from individual patients, and to implicate specific C. auris genes and gene variants in echinocandin tolerance and resistance, and in virulence. In aim 1, we will perform short- and long- read whole genome sequencing and establish phylogeny of multiple clade I and III C. auris recovered from cultures from each patient, including at baseline and during persistent and recurrent infections despite echinocandin treatment. We will then test phenotypes of genetically distinct strains from each patient, including echinocandin tolerance and resistance, and virulence-associated phenotypes. In aim 2, we will prioritize genes and gene variants, and create isogenic mutant C. auris strains in clade I and III backgrounds. Isogenic strains will be assessed for echinocandin responses and virulence in vitro and in a mouse model of bloodstream infection. Results will afford new insights into echinocandin responses and pathogenesis by C. auris, and identify genes that contribute to these processes. Our results will provide a foundation for mechanistic studies of echinocandin tolerance, resistance and virulence, and for trials establishing the clinical significance of C. auris genotypic...