RNAi-dependent epimutation roles in antimicrobial drug resistance and pathogenesis

抽象的。本提案重点关注人类真菌致病毛霉菌种复合体，一组相关 导致难以治疗的毁灭性感染的病原体，药物治疗选择有限， 在某些患者中需要外科清创术。在过去的十年里，我们和其他人推进了基因组学， 遗传学和动物模型，为这一未充分研究的微生物病原体组。我们发现这种蛋白质 磷酸酶钙调神经磷酸酶控制毛霉致病所需的从酵母到菌丝的二态转变， 并通过对FK506耐药菌株的研究发现了一种新的抗菌药物耐药机制。 在以前发表的和初步的研究中，通过我们的发现， 一种称为表突变的抗真菌药物耐药性的新机制， 并使药物靶基因沉默。该途径赋予短暂、不稳定的耐药性和耐药分离株 在没有药物的情况下迅速恢复药物敏感性。通过基因和分子研究，我们定义了 差向突变所需的RNAi组分、差向突变所需的RNAi组分和新的类别 抑制表型突变的形成通过表位突变介导的抗菌药物耐药性的发现 已经概括了：1）显示表突变发生在两种不同的致病性毛霉属，2）定义了一个 控制表突变频率和稳定性的替代RNAi途径，3）在另外的RNAi途径中鉴定表突变， 引起抗真菌剂抗性的基因，以及4）记录了表突变在动物体内持续存在， 感染或在动物传代后出现。这些见解为本文提出的研究奠定了基础， 表位突变的机制，并阐明表位突变对微生物病原体相互作用的影响， 主持人在目前的建议中，我们假设表突变是一个普遍的过程，在许多 真核微生物病原体，并作为一个主要的力量，在抗菌药物耐药性，控制目标 涉及药物作用、基因组稳定性和真核微生物病原体发病机理的基因。 我们的研究将揭示RNAi导致表位突变的独特方面，表位突变介导抗菌药物 人类普遍存在的真菌病原体的耐药性。目的1：1）阐明 表位突变和靶点，包括涉及耐药性的基因（包括临床使用的抗真菌药物） 和转座因子，以及它们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，这可能会导致表位突变的出现，以及3）建立普遍性 将这些发现应用于其他致病真菌物种。目标2将定义表突变对抗菌药物的影响 微生物与免疫细胞、血脑屏障、类器官 和全动物模型。这些研究将促进我们对抗菌药物耐药性的理解， 可以通过一种新的基于RNAi的途径进化，对感染性疾病的进化，治疗， 和预防，并提供对其他真核病原体与活性RNAi途径的见解。

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.