Mapping the genomic and molecular mechanisms of antifungal resistance in the emerging fungal pathogen Candida auris

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PROJECT SUMMARY/ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen of great clinical concern that is associated with outbreaks on six continents. A critical barrier to overcoming the high antifungal resistance in C. auris is the significant lack of understanding of its genetic, genomic, and molecular basis. The long-term goal of this project is to advance the treatment of C. auris. The overall objective of this proposal is to fully understand the molecular and genetic basis of antifungal resistance in clinical isolates of this fungal pathogen. The central hypothesis behind this proposal is that antifungal resistance is mediated both by mechanisms analogous to those previously identified in other species of Candida, as well as novel mechanisms unique to this organism. Aim 1 is to identify and delineate the genetic determinants of triazole resistance in clinical isolates of C. auris. Preliminary studies identified mutations in genes encoding the triazole target sterol demethylase (Erg11) and the novel transcription factor (TF) Tac1B as contributing to resistance in some but not all isolates. Genome Wide Association Studies (GWAS) and transcriptional profiling of a growing collection of clinical and experimentally evolved isolates will be used to identify novel genes and mutations linked to triazole resistance which will be tested using CRISPR- Cas9 gene editing. The contribution of activating mutations in TF genes such as TAC1B and MRR1A will be determined using RNA-seq and ChIP-seq. Aim 2 is to Identify and delineate the genetic determinants of echinocandin resistance in clinical isolates of C. auris. Mutations in the gene encoding glucan synthase (FKS1) have been identified among isolates resistant to echinocandins. However, preliminary data indicate that some resistant isolates lack FKS1 mutations and others exhibit high-level resistance that cannot be explained by such mutations alone. CRISPR-Cas9 gene editing will be used to assess mutations in FKS1 for their individual contribution to resistance. Novel candidate resistance genes and mutations will be identified using GWAS and transcriptional profiling of clinical and experimentally evolved isolates and these genes will be tested by CRISPR- Cas9 gene editing. Aim 3 is to identify and delineate the genetic determinants of amphotericin B resistance in clinical isolates of C. auris. In preliminary studies, mutations in the gene encoding sterol methyltransferase (Erg6) were identified as a cause of resistance to amphotericin B. Novel resistance genes and mutations will be identified using comprehensive sterol profiling, GWAS, and transcriptional profiling of clinical and experimentally evolved isolates, and mutations will be tested by CRISPR-Cas9 gene editing. The relationship between resistance and fitness will also be determined. Understanding the basis of antifungal resistance is essential for the development of tools to better predict response to therapy, to aid rational drug design and development, and to assist in epidemiologic tracking of the spread of resistance. The proposed project will, for the first time, undertake a comprehensive examination of the genomic, genetic, and molecular basis of antifungal resistance in the emerging multi-drug resistant fungal pathogen C. auris.