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

项目总结/摘要 耳念珠菌是一种新出现的多重耐药真菌病原体，临床上备受关注， 六大洲爆发疫情目前，克服念珠菌高耐药性的一个关键障碍是：奥里斯是 严重缺乏对其遗传、基因组和分子基础的了解。这个项目的长期目标是 是推进C.耳。本提案的总体目标是充分了解分子 以及该真菌临床分离株耐药的遗传基础。核心假设 这一提议背后的原因是，抗真菌耐药性是由与先前类似的机制介导的， 在念珠菌的其他物种中鉴定，以及这种生物体特有的新机制。目标1：识别 并描述了临床分离的念珠菌三唑耐药的遗传决定因素。耳。初步研究 在编码三唑靶固醇脱甲基酶（Erg 11）的基因中发现了突变， 因子（TF）Tac 1B在一些但不是所有分离株中导致耐药性。全基因组关联研究 （GWAS）和转录谱的临床和实验进化的分离物越来越多的集合将 用于鉴定与三唑耐药性相关的新基因和突变，这些基因和突变将使用CRISPR- Cas9基因编辑。TF基因如TAC 1B和MRR 1A中激活突变的贡献将是 使用RNA-seq和ChIP-seq测定。目的2是确定和描述遗传决定因素， 棘白菌素耐药的临床分离的C.耳。编码葡聚糖合酶（FKS 1）的基因突变 已经在棘白菌素耐药菌株中鉴定出。然而，初步数据显示， 耐药菌株缺乏FKS 1突变，其他菌株表现出高水平的耐药性，这不能用这种突变来解释。 只有变异CRISPR-Cas9基因编辑将用于评估FKS 1中的突变， 对抵抗的贡献。新的候选耐药基因和突变将使用GWAS鉴定， 临床和实验进化的分离株的转录谱分析，这些基因将通过CRISPR- Cas9基因编辑。目的3是鉴定和描述大肠埃希氏菌对阿替霉素B耐药的遗传决定因素， 临床分离的C.耳。在初步研究中，编码甾醇甲基转移酶（Erg 6）的基因突变 被鉴定为对阿替霉素B耐药的原因。新的耐药基因和突变将是 使用全面的固醇分析，GWAS和临床和实验的转录谱鉴定， 进化的分离株和突变将通过CRISPR-Cas9基因编辑进行测试。的关系 还将确定阻力和适应性。了解抗真菌药物耐药性的基础对于 开发更好地预测治疗反应的工具，以帮助合理的药物设计和开发，以及 以协助对耐药性传播的流行病学追踪。该项目将首次 对抗真菌药物耐药性的基因组、遗传和分子基础进行全面检查 在新出现的多重耐药真菌病原体C.耳。

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.