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.

烟曲霉克服三唑耐药性的一个关键障碍是严重缺乏 了解其遗传和分子基础。我们已经表明，已知的耐药机制确实 不能完全解释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 治疗并确保三唑类药物在治疗由以下原因引起的感染方面的持续临床效用 曲霉属物种。我们的中心假设是非 cyp51A 突变介导的机制是必不可少的 烟曲霉临床分离株对三唑耐药，涉及复杂的遗传变化，改变 1) 甾醇 生物合成及其转录激活，2) 三唑转运及其转录激活，以及 3) 目前为止 未知的机制。我们当前的目标是通过识别遗传因素来解决关键的知识差距 和非 cyp51A 突变介导的耐药性的分子决定因素。我们的初步数据表明 虽然 cyp51A 突变在三唑耐药临床分离株中很常见，但它们对 阻力很小。我们观察到了我们收集的抗性分离株特有的基因突变 编码甾醇传感蛋白、甾醇生物合成调节剂和甾醇生物合成酶。我们有 还观察到临床分离株不仅过度表达 cyp51A，而且过度表达麦角甾醇生物合成的大多数基因 途径，表明其组成型激活。我们观察到几个潜在的转运蛋白正在上升- 在我们收集的三唑耐药分离株中受到调节，表明三唑外流和耐药性的作用 由这些运输商。我们还表明，烟曲霉的临床分离株通过吸收三唑类抗真菌药物 促进扩散，我们认为改变三唑进口可能代表了一种重要的机制 反抗。为了实现我们的目标，我们将进行实验来了解什么 遗传和分子决定因素通过改变甾醇生物合成或其代谢影响三唑敏感性 转录激活（目标 1）和三唑转运及其调节（目标 2）。在目标 3 中，我们还将利用 无偏见的全基因组比较，加上体外进化实验，以确定完全新颖的 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遗传和 基因组技术研究和发现新的非 cyp51A 突变介导的三唑机制 在美国的三唑耐药临床分离株中发挥作用的耐药性。拟议的 研究意义重大，因为它代表了对非遗传性疾病的分子和遗传基础的全面分析。 cyp51A 突变介导烟曲霉的三唑抗性，并将提供新的见解 可以提高三唑对这种重要人类病原体的活性。