RNAi-dependent epimutation roles in antimicrobial drug resistance and pathogenesis

抽象的。这项建议主要针对人类真菌致病毛霉物种复合体、一组相关的 导致难以治疗的破坏性感染的病原体，药物治疗选择有限，以及 在一些病人中需要手术清创。在过去的十年里，我们和其他人发展了基因组学， 遗传学，以及这组未被研究的微生物病原体的动物模型。我们发现了这种蛋白质 磷酸酶钙调神经磷酸酶控制毛霉发病所需的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素耐药机制。 在之前发表的和初步的研究中，我们的发现取得了重大进展 一种新的抗真菌耐药机制，称为表亲和作用，通过它激活RNAi途径 并使药物靶标基因沉默。这种途径使暂时的、不稳定的耐药和耐药分离株。 在没有药物的情况下，迅速恢复药物敏感性。通过遗传和分子研究，我们定义了 变异所需的RNAi成分、变异所必需的RNAi成分和一种新的类别 这抑制了表型突变的形成。表型突变介导的抗菌素耐药性的发现 已被概括为：1)显示突变发生在两个不同的致病毛霉物种中，2)定义一个 控制突变频率和稳定性的替代RNAi途径，3)识别附加的突变 导致对抗真菌药物产生抗药性的基因，以及4)证明在动物体内持续的表观突变 感染或在动物传代后出现。这些见解为这里提出的研究奠定了基础，以进一步定义 以及阐明突变对病原菌与微生物相互作用的影响 主持人。在目前的提案中，我们假设置换是一个普遍的过程，它跨越许多 真核微生物病原体，是控制靶标的抗菌素耐药性的主要力量 涉及真核微生物病原体的药物作用、基因组稳定性和致病机制的基因。 我们的研究将揭示RNAi导致表位突变的独特方面，而表位突变是介导抗微生物药物的 人类普遍存在的真菌病原体的耐药性。目的1)阐明其分子机制 突变和靶点，包括与耐药有关的基因(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，这可能会推动表型突变的出现，以及3)建立概括性 将这些发现推广到其他病原真菌物种。目标2将定义突变对抗菌素的影响 微生物与免疫细胞、血脑屏障、有机物、 和全动物模型。这些研究将增进我们对抗菌素耐药性的理解 可以通过一种新的基于RNAi的途径进化，对传染病的进化、治疗、 和预防，并提供了对具有活跃的RNAi途径的其他真核病原体的见解。

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.