Structural and Functional Characterization of RNA polymerase and its Regulators from Mycobacterium tuberculosis and Clostridioides difficile

项目摘要 由结核分枝杆菌(Mtb)引起的结核病(TB)仍然是一个主要的健康问题。 疾病控制中心估计，全球约有三分之一至四分之一的人口存在潜伏期 被感染了。RNA聚合酶(RNAP)是负责细菌中所有转录的酶，是 利福霉素(Rif)类抗生素，结核病的一线治疗方法。因此，RNAP是经过验证的 是开发新药的有吸引力的目标。这突显了我们最近的结构和 Mtb RNAP的功能特征及其两个必需转录因子在FULL中的作用 转录活性。上一次的资助使我们能够提供2.8？分辨率的晶体结构 耻垢分枝杆菌的RNAP转录起始复合体(TIC)和结核分枝杆菌最近的冷冻-EM结构 转录复合体。在这项提议中，冷冻-EM将被用来检查RNAP复合体作为起点 为了阐明一个相对未知的转录因子家族的机制，WhiB因子。 WhiB因子只在放线杆菌中发现，并在结核分枝杆菌中发挥作用，包括对生长和 分裂和对宿主诱导的应激反应，包括抗生素耐受性，一氧化氮，巨噬细胞 入侵和活性氧物种。我们将使用多学科方法，包括结构， 生物化学、基因组和活体实验(与J.Rock合作)以了解角色和 这一重要但相对未知的转录因子家族的机制。结果来自于 AIMS在这里不仅有可能阐明这些因素的机制和生物学，而且还提供 为特定支系抗生素开发的新目标提供了一个平台，并指导我们如何增加 目前的抗生素配方的有效性。 上一次资助期间的结果导致了分枝杆菌RNAP的高分辨率结构(由 冷冻-EM和结晶学)，并提供了表征Rif和Rif如何衍生的机会 抑制Rif耐药(RIFR)细菌抑制分枝杆菌RNAP。在这里，我们建议继续这条研究路线 具有结构上未表征的Rif衍生物，由S.Brady提供，抑制额外的RIFR Mtb RNAP。 艰难梭状芽胞杆菌是一种革兰氏阳性、产孢子菌、厌氧菌，是一种条件致病菌 这对受威胁的主机来说是致命的。非达索米星(FDX)，FDA批准的唯一其他针对 RNAP，是治疗Cdiff感染的有效药物。我们最近的工作证实，FDX可以抑制Mtb RNAP 是有效的，但这种效力取决于放线杆菌特异的转录因子RpbA，它是 在Cdiff中缺席。在这里，我们建议扩展我们在细菌的生化和结构研究方面的专业知识 RNAP包括Cdiff所属的以前未被描述的Firmiuts支系。这里的结果是 将阐明FDX效力的结构和生化基础，并提供结构和 利用Cdiff RNAP进行药物开发和优化的生化基础。

Project Summary Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to pose a major health problem. The Center for Disease Control estimates that approximately 1/3 to 1/4 of the world’s population is latently infected. RNA polymerase (RNAP), the enzyme responsible for all transcription in bacteria, is the target for the Rifamycin (Rif) class of antibiotics, a first line therapeutic treatment for TB. RNAP is thus a proven and attractive target for the development of new drugs. This highlights the importance of our recent structural and functional characterization of Mtb RNAP and the roles of two essential transcription factors required for full transcriptional activity. The previous grant enabled us to provide a 2.8 Å resolution crystal structure of an RNAP transcription initiation complex (TIC) from M. smegmatis and more recently cryo-EM structures of Mtb transcription complexes. In this proposal, cryo-EM will be used to examine RNAP complexes as a starting point to elucidate the mechanisms of a family of relatively uncharacterized transcription factors, the WhiB factors. The WhiB factors are only found in Actinobacteria and have roles in Mtb that include essentiality for growth and division, and responses to host induced stresses including antibiotic tolerance, nitric oxide, macrophage invasion and reactive oxygen species. We will use a multidisciplinary approach that includes structural, biochemical, genomic and in vivo experiments (in collaboration with J. Rock) to understand the roles and mechanism of this important, but relatively uncharacterized family of transcription factors. The results from the aims here have the potential to not only elucidate the mechanism and biology of these factors, but also provide a platform for new targets for clade-specific antibiotic development and serve to guide us on how to increase the efficacy of the current repertoire of antibiotics. The results from the previous funding period have led to high resolution structures of Mycobacteria RNAP (by cryo-EM and crystallography), and provided the opportunity to characterize how Rif and Rif derivatives that inhibit Rif resistant (RifR) bacteria inhibit Mycobacteria RNAP. Here we propose to continue this line of research with structurally uncharacterized Rif derivatives, provided by S. Brady, that inhibit additional RifR Mtb RNAPs. Clostrioides difficile (Cdiff), a Gram-positive, sporulating, anaerobic bacterium, is an opportunistic pathogen which is deadly to compromised hosts. Fidaxomicin (Fdx), the only other FDA approved antibiotic which targets RNAP, is a powerful treatment for Cdiff infection. Our recent work established that Fdx can inhibit Mtb RNAP potently, but that potency is dependent on the Actinobacteria-specific transcription factor RpbA, which is absent in Cdiff. Here we propose to extend our expertise in biochemical and structural studies of bacterial RNAPs to include the previously uncharacterized clade of Firmicutes to which Cdiff belongs. The results here will elucidate the structural and biochemical basis for Fdx potency as well as provide a structural and biochemical basis for exploiting Cdiff RNAP for drug development and optimization.