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

绘制新兴真菌病原体耳念珠菌抗真菌耐药性的基因组和分子机制

• 批准号: 10587792
• 负责人: Christina A Cuomo
• 金额: $ 124.79万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-06 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10587792
• 关键词: Affect; Amphotericin B; Antifungal Agents; Antimicrobial Resistance; Azole resistance; Azoles; CRISPR/Cas technology; Candida; Candida auris; Candidiasis; Cells; Centers for Disease Control and Prevention (U.S.); ChIP-seq; Clinical; Collection; Data; Development; Diagnostic; Disease Outbreaks; Drug Design; Drug resistance; Epidemiology; Exhibits; Fluconazole resistance; Fungal Drug Resistance; Gene Expression Profiling; Genes; Genetic; Genetic Determinism; Genomics; Goals; Individual; Infection; Knowledge; Link; Maps; Mediating; Methyltransferase; Molecular; Morbidity - disease rate; Multi-Drug Resistance; Multiple Fungal Drug Resistance; Mutation; Organism; Outcome; Prediction of Response to Therapy; Public Health; Publishing; Reporting; Resistance; Resistance development; Sterols; Testing; Therapeutic; Time; Translating; United States; Work; candidate identification; combat; cost; drug development; echinocandin resistance; emerging pathogen; fitness; genome wide association study; glucan synthase; mortality; multi-drug resistant pathogen; novel; pathogenic fungus; public health relevance; resistance gene; resistance mechanism; resistance mutation; time use; tool development; transcription factor; transcriptome sequencing

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.

项目概要/摘要 耳念珠菌是一种新兴的多重耐药真菌病原体，引起临床高度关注，与 六大洲爆发疫情。克服耳念珠菌高抗真菌耐药性的一个关键障碍是 对其遗传、基因组和分子基础严重缺乏了解。本项目的长期目标 是为了推进耳念珠菌的治疗。该提案的总体目标是充分了解分子 该真菌病原体临床分离株的抗真菌耐药性和遗传基础。中心假设 这一提议的背后是抗真菌耐药性是通过类似于之前的机制来介导的 在其他念珠菌物种中发现的，以及该生物体独特的新机制。目标 1 是确定 并描述耳念珠菌临床分离株三唑耐药性的遗传决定因素。初步研究 鉴定出编码三唑靶甾醇脱甲基酶（Erg11）的基因突变和新的转录 因子 (TF) Tac1B 导致某些但不是所有分离株产生耐药性。全基因组关联研究 （GWAS）和越来越多的临床和实验进化分离株的转录谱分析将 用于识别与三唑耐药性相关的新基因和突变，并将使用 CRISPR 进行测试 Cas9基因编辑。激活 TF 基因突变（例如 TAC1B 和 MRR1A）的贡献将是 使用 RNA-seq 和 ChIP-seq 确定。目标 2 是识别和描述遗传决定因素 耳念珠菌临床分离株中的棘白菌素耐药性。编码葡聚糖合酶 (FKS1) 的基因突变 已鉴定出对棘白菌素具有抗性的分离株。然而，初步数据表明，一些 耐药菌株缺乏 FKS1 突变，而其他菌株则表现出高水平的耐药性，但无法用这种突变来解释 单独突变。 CRISPR-Cas9基因编辑将用于评估其个体的FKS1突变 抵抗力的贡献。新的候选耐药基因和突变将使用 GWAS 进行鉴定， 临床和实验进化分离株的转录谱分析，这些基因将通过 CRISPR 进行测试 Cas9基因编辑。目标 3 是确定和描述两性霉素 B 耐药性的遗传决定因素 耳念珠菌的临床分离株。在初步研究中，编码甾醇甲基转移酶（Erg6）的基因发生突变 被确定为两性霉素 B 耐药性的一个原因。新的耐药基因和突变将被 使用综合甾醇分析、GWAS 以及临床和实验的转录分析进行鉴定 进化分离物，突变将通过 CRISPR-Cas9 基因编辑进行测试。之间的关系 抵抗力和健康状况也将被确定。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 协助流行病学追踪耐药性的传播。拟议的项目将首次 对抗真菌耐药性的基因组、遗传和分子基础进行全面检查 在新兴的多重耐药真菌病原体耳念珠菌中。