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

项目摘要/摘要 金黄色念珠菌是一种新出现的临床高度关注的多重耐药真菌病原菌，与 在六大洲爆发疫情。克服金黄色葡萄球菌高抗药性的一个关键障碍是 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 是推进对金黄色葡萄球菌的治疗。这项提议的总体目标是充分理解分子 以及该病原菌临床分离株耐药的遗传基础。中心假说 这一提议背后的原因是，抗真菌耐药性是通过与以前类似的机制来调节的 在假丝酵母菌的其他物种中发现，以及这种生物特有的新机制。目标1是确定 并描述了临床分离的金黄色葡萄球菌对三氮唑耐药性的遗传决定因素。初步研究 三氮唑靶向固醇去甲基酶(Erg11)编码基因的突变和新的转录 因子(Tf)Tac1B在一些但不是所有分离物中起到抗药性的作用。全基因组关联研究 对越来越多的临床和实验进化的分离株进行(Gwas)和转录图谱分析将 用于识别与三氮唑耐药性相关的新基因和突变，将使用CRISPR- Cas9基因编辑。TAC1B和MRR1A等TF基因的激活突变的贡献将是 用RNA-SEQ和CHIP-SEQ测定。目标2是确定和描述人类免疫缺陷病毒的遗传决定因素 金黄色葡萄球菌临床分离株对棘球绦虫的耐药性葡聚糖合成酶(FKS1)编码基因突变 在对棘球菌素类药物耐药的菌株中已被鉴定。然而，初步数据表明，一些人 耐药菌株缺乏FKS1突变，其他菌株表现出无法用这种方式解释的高水平耐药性 仅突变一项。CRISPR-Cas9基因编辑将用于评估他们个人的FKS1突变 对抵抗的贡献。新的候选抗病基因和突变将利用GWAs和 临床和实验进化分离株的转录图谱，这些基因将通过CRISPR- Cas9基因编辑。目的3鉴定和描述鸡对两性霉素B抗性的遗传决定因素。 金黄色葡萄球菌临床分离株。在初步研究中，编码甾醇甲基转移酶(Erg6)的基因突变 被确定为对两性霉素B产生耐药性的原因。新的耐药性基因和突变 使用临床和实验的全面的甾醇图谱、GWAS和转录图谱进行鉴定 进化的分离株和突变将通过CRISPR-Cas9基因编辑进行测试。两国之间的关系 抵抗力和体能也将被确定。了解抗真菌耐药性的基础对于 开发工具以更好地预测治疗反应，帮助合理的药物设计和开发，以及 协助对耐药性传播的流行病学跟踪。拟议中的项目将首次， 对抗真菌耐药性的基因组、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金黄色葡萄球菌中。

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.