Non-cyp51A-mutation Mediated Triazole Resistance in Aspergillus fumigatus

非 cyp51A 突变介导的烟曲霉三唑耐药性

• 批准号: 10582526
• 负责人: Jarrod R. Fortwendel
• 金额: $ 65.73万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582526
• 关键词: 3-hydroxy-3-methylglutaryl-coenzyme A; Address; Affect; Alleles; Anabolism; Antifungal Agents; Antifungal Therapy; Aspergillosis; Aspergillus; Aspergillus fumigatus; Automobile Driving; Azole resistance; Azoles; CRISPR/Cas technology; Clinical; Collection; Complex; Coupled; DNA Sequence Alteration; Data; Diagnosis; Diffusion; Disease; Drug Transport; Ensure; Ergosterol; Evolution; Exhibits; Fungal Drug Resistance; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic; Genome; Genomics; Goals; Immunocompromised Host; In Vitro; Individual; Infection; Knowledge; Laboratories; Mediating; Methods; Molds; Molecular; Molecular Analysis; Morbidity - disease rate; Mutation; Outcome; Oxidoreductase; Pathway interactions; Play; Predisposition; Proteins; Regulation; Research; Resistance; Resistance development; Resistance profile; Risk; Role; Sequence Analysis; Sterol Biosynthesis Pathway; Sterols; System; Techniques; Testing; Therapeutic; Transcriptional Activation; Translating; Treatment Failure; Voriconazole; Work; clinical predictors; clinically relevant; differential expression; effective therapy; enzyme biosynthesis; experimental study; genetic analysis; genetic manipulation; genome analysis; human pathogen; improved; inhibitor; innovation; insight; mortality; mutant; novel; novel strategies; overexpression; patient population; prevent; public health relevance; resistance mechanism; resistance mutation; resistant Aspergillus; stem; treatment choice; whole genome

A critical barrier to overcoming triazole resistance in Aspergillus fumigatus is the significant lack of understanding of its genetic and molecular basis. We have shown that the known mechanisms of resistance do not fully explain resistance observed among most clinical isolates. Our long-term goal is to improve antifungal therapy and ensure the sustained clinical utility of the triazole class for treatment of infections caused by Aspergillus species. Our central hypothesis is that non-cyp51A-mutation mediated mechanisms are essential to triazole resistance in clinical isolates of A. fumigatus and involve complex genetic changes altering 1) sterol biosynthesis and its transcriptional activation, 2) triazole transport and its transcriptional activation, and 3) as yet unknown mechanisms. Our current objective is to address critical knowledge gaps by identifying the genetic and molecular determinants of non-cyp51A-mutation mediated resistance. Our preliminary data suggest that while mutations in cyp51A among triazole resistant clinical isolates are common, their overall contribution to resistance is minimal. We have observed mutations, unique to resistant isolates in our collection, in genes encoding sterol sensing proteins, regulators of sterol biosynthesis, and sterol biosynthesis enzymes. We have also observed clinical isolates that overexpress not only cyp51A, but most genes of the ergosterol biosynthesis pathway, suggesting its constitutive activation. We have observed several potential transporters that are up- regulated among triazole resistant isolates in our collection, suggesting a role for triazole efflux and resistance by these transporters. We have also shown that clinical isolates of A. fumigatus take up triazole antifungals via facilitated diffusion and we believe that altered triazole import may represent an important mechanism of resistance. To accomplish our objective we will undertake experiments that will lead to an understanding of what genetic and molecular determinants influence triazole susceptibility through altered sterol biosynthesis or its transcriptional activation (Aim 1) and triazole transport and its regulation (Aim 2). In Aim 3, we will also utilize an unbiased whole genome comparisons, coupled with in vitro evolution experiments, to identify completely novel mechanisms of resistance in clinical isolates. Our approach is innovative as we will use the latest genetic and genomic techniques to study and discover novel non-cyp51A-mutation mediated mechanisms of triazole resistance that are operative in a U.S.-based collection of triazole resistant clinical isolates. The proposed research is significant as it represents a comprehensive analysis of the molecular and genetic basis of non- cyp51A-mutation mediated triazole resistance in A. fumigatus and will provide novel insights into ways in which triazole activity can be improved against this important human pathogen.

克服烟曲霉对三氮唑耐药性的一个关键障碍是显著缺乏 了解其遗传和分子基础。我们已经证明，已知的抗药性机制确实 不能完全解释在大多数临床分离株中观察到的耐药性。我们的长期目标是提高抗真菌能力 治疗并确保三氮唑类药物治疗由以下原因引起的感染 曲霉菌种。我们的中心假设是非cyp51a突变机制是必不可少的。 临床分离烟曲霉菌对三氮唑的耐药性和涉及改变1)甾醇的复杂基因变化 生物合成及其转录激活，2)三氮唑转运及其转录激活，3)尚未 未知的机制。我们目前的目标是通过识别基因，解决关键的知识差距 以及非cyp51a突变介导的抗性的分子决定因素。我们的初步数据表明 虽然cyp51a基因突变在对三氮唑耐药的临床分离株中很常见，但它们对 阻力是最小的。我们观察到了我们收集的耐药菌株所特有的基因突变。 编码类固醇感应蛋白、类固醇生物合成调节剂和类固醇生物合成酶。我们有 还观察到临床分离株不仅过度表达cyp51a，而且过度表达麦角固醇生物合成的大部分基因。 途径，提示其结构性激活。我们观察到了几个潜在的传输器正在运行- 在我们收集的三氮唑耐药分离株中进行调节，提示三氮唑外排和耐药性的作用 被这些传送者。我们还发现，烟曲霉菌的临床分离株通过 促进了扩散，我们认为改变的三氮唑进口可能代表了 抵抗。为了实现我们的目标，我们将进行实验，以了解 基因和分子决定因素通过改变甾醇的生物合成或改变其对三氮唑的敏感性而影响其敏感性 转录激活(目标1)和三氮唑转运及其调控(目标2)。在目标3中，我们还将利用 无偏见的全基因组比较，结合体外进化实验，以确定完全新的 临床分离株的耐药机制。我们的方法是创新的，因为我们将使用最新的基因和 基因组技术研究和发现新的非cyp51a突变介导的三氮唑作用机制 在美国的三氮唑耐药临床分离株集合中起作用的耐药性。建议数 这项研究具有重要意义，因为它代表了对非传染性疾病的分子和遗传学基础的全面分析 Cyp51a突变介导了烟曲霉菌对三氮唑的耐药性，并将为研究 三氮唑对这种重要的人类病原体的活性可以提高。