RNAi-dependent epimutation roles in antimicrobial drug resistance and pathogenesis

RNAi 依赖性表突变在抗菌药物耐药性和发病机制中的作用

• 批准号: 10654857
• 负责人: JOSEPH HEITMAN
• 金额: $ 74.23万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10654857
• 关键词: Animal Model; Animals; Antifungal Agents; Antisense RNA; Azoles; Blood - brain barrier anatomy; Brain; Categories; Cells; Central Nervous System Infections; Class Zygomycetes; Clinical; Coculture Techniques; Complex; DNA Transposable Elements; Debridement; Development; Distant; Drug Targeting; Drug resistance; Drug resistance pathway; Engineering; Evolution; Exhibits; FK506; Foundations; Frequencies; Fungal Drug Resistance; Gene Silencing; Genes; Genetic; Genetic Models; Genome Stability; Genomics; Germination; Helicase Gene; Human; Hyphae; Immune; Immunocompetent; Immunocompromised Host; In Vitro; Infection; Link; Macrophage; Mating Types; Mediating; Messenger RNA; Microbe; Microbial Drug Resistance; Modeling; Molecular; Morbidity - disease rate; Mucor; Mucormycosis; Operative Surgical Procedures; Organ; Organoids; Outcome Study; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phylogenetic Analysis; Polyenes; Positioning Attribute; Prevention; Prevention strategy; Process; Protein phosphatase; Publishing; RNA Helicase; RNA Interference; Resistance; Rhizopus; Role; Series; Sexual Reproduction; Small RNA; Stress; Testing; Yeasts; calcineurin phosphatase; drug action; drug sensitivity; drug-sensitive; genome-wide; host-microbe interactions; human pathogen; in vitro Model; infectious disease evolution; insight; microbial; mortality; novel; pathogen; pathogenic fungus; pathogenic microbe; response; transcriptome; transcriptome sequencing; treatment strategy

Abstract. This proposal focuses on the human fungal pathogenic Mucor species complex, a group of related pathogens that cause devastating infections that are difficult to treat, with limited drug treatment options, and requiring surgical debridement in some patients. Over the past decade, we and others have advanced genomics, genetics, and animal models for this understudied group of microbial pathogens. We discovered the protein phosphatase calcineurin controls the dimorphic transition from yeast to hyphae required for Mucor pathogenesis, and through studies of FK506-resistant isolates discovered a novel mechanism of antimicrobial drug resistance. In previously published and preliminary studies, significant advances were achieved through our discovery of a novel mechanism of antifungal drug resistance called epimutation, whereby the RNAi pathway is activated and silences drug target genes. This pathway confers transient, unstable drug resistance, and resistant isolates rapidly revert to drug sensitivity in the absence of drug. Through genetic and molecular studies, we defined RNAi components required for epimutation, those that are dispensable for epimutation, and a novel category that inhibits formation of epimutations. The discovery of antimicrobial drug resistance mediated via epimutations has been generalized: 1) showing epimutation occurs in two different pathogenic Mucor species, 2) defining an alternative RNAi pathway controlling epimutation frequency and stability, 3) identifying epimutations in additional genes causing resistance to antifungal agents, and 4) documenting that epimutations persist during animal infection or arise after animal passage. These insights set the stage for studies proposed here to further define mechanisms of epimutation, and elucidate the impact of epimutations on microbial pathogen interactions with the host. In the current proposal, we hypothesize epimutation is a general process that operates across many eukaryotic microbial pathogens, and acts as a major force in antimicrobial drug resistance that controls target genes involved in drug action, genome stability, and pathogenesis of eukaryotic microbial pathogens. Our studies will reveal unique facets of RNAi that lead to epimutations, which mediate antimicrobial drug resistance in ubiquitous fungal pathogens of humans. Aim 1 will 1) elucidate molecular mechanisms of epimutation and targets, including genes involved in drug resistance (including clinically used antifungal drugs) and transposable elements, and their impact on genome stability, 2) define conditions, including stress, sexual reproduction, and infection, that may drive the emergence of epimutations, and 3) establish the generalizability of these findings to other pathogenic fungal species. Aim 2 will define the impact of epimutation on antimicrobial drug resistance and pathogenicity in microbe interactions with immune cells, the blood-brain barrier, organoids, and whole-animal models. These studies will advance our understanding of how antimicrobial drug resistance can evolve via a novel RNAi-based pathway with direct implications for infectious disease evolution, treatment, and prevention, and provide insights into other eukaryotic pathogens with active RNAi pathways.

抽象的。该提案重点是人类真菌致病性粘液物种复合物，这是一组相关的 引起毁灭性感染的病原体，具有有限的药物治疗选择，并且 在某些患者中需要手术清创术。在过去的十年中，我们和其他人拥有先进的基因组学， 该研究的微生物病原体的遗传学和动物模型。我们发现了蛋白质 磷酸酶钙调蛋白控制粘液发病机理所需的酵母二态过渡， 通过研究FK506抗性分离株，发现了一种新型的抗菌耐药性机制。 在先前发表的初步研究中，通过我们的发现取得了重大进步 一种称为Epimount的新型抗真菌耐药性的机制，从而激活RNAi途径 和沉默药物靶基因。该途径赋予了瞬态，不稳定的耐药性和抗性分离株 在没有药物的情况下，迅速恢复为药物敏感性。通过遗传和分子研究，我们定义了 epimunion所需的RNAi组件，可用于估算的rnai组件以及新型类别 这抑制了表述的形成。发现通过日期介导的抗菌耐药性的发现 已被概括：1）显示在两个不同的致病性粘液物种中发生脱位，2）定义 控制释放频率和稳定性的替代RNAi途径，3）识别额外的缩影 引起对抗真菌剂的耐药性的基因，4）记录在动物期间的表述持续存在 动物通道后感染或出现。这些见解为这里提出的研究奠定了阶段，以进一步定义 释放的机制，并阐明了扩展对微生物病原体相互作用与与 主人。在当前的建议中，我们假设夸张是一个一般过程，在许多过程中运作 真核生物微生物病原体，充当控制靶标的抗菌耐药性的主要力 与真核微生物病原体的药物作用，基因组稳定性和发病机理有关的基因。 我们的研究将揭示RNAi的独特方面，导致表达，从而介导抗菌药物 人类无处不在的真菌病原体的抗性。 AIM 1将1）阐明分子机制 取代和靶标，包括涉及耐药性的基因（包括临床使用的抗真菌药物） 和转座元素及其对基因组稳定性的影响，2）定义条件，包括压力，性 繁殖和感染，可能会推动表演的出现，3）建立普遍性 这些发现是其他致病真菌物种的。 AIM 2将定义释放对抗微生物的影响 微生物相互作用的耐药性和致病性与免疫细胞，血脑屏障，类器官， 和全动物模型。这些研究将提高我们对抗菌药物抗性的理解 可以通过一种新型的基于RNAi的途径进化，对感染疾病进化，治疗，直接影响 和预防，并通过活性RNAi途径向其他真核病原体提供见解。