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）继续构成主要的健康问题。 疾病控制中心估计，世界上大约1/3到1/4的人口是潜伏性的。 感染了RNA聚合酶（RNAP）是细菌中负责所有转录的酶，是RNA聚合酶的靶标。 利福霉素（Rif）类抗生素，结核病的一线治疗药物。因此，RNAP是一种经过验证的， 这是开发新药的一个有吸引力的目标。这突出了我们最近的结构和 结核分枝杆菌RNAP的功能特征和两个必需的转录因子的作用，需要充分的 转录活性先前的资助使我们能够提供一个2.8纳米分辨率的晶体结构， RNAP转录起始复合物（TIC）来自M.结核分枝杆菌的涂片和最近的冷冻电镜结构 转录复合体在这个建议中，cryo-EM将被用来检查RNAP复合物作为一个起点 阐明一个家族的相对不典型的转录因子，WhiB因子的机制。 WhiB因子仅在放线菌中发现，并且在Mtb中具有包括生长必需性和 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。我们将采用多学科方法，包括结构， 生物化学，基因组和体内实验（与J. Rock合作），以了解其作用， 这个重要的，但相对不典型的转录因子家族的机制。的结果 这里的目的不仅有可能阐明这些因素的机制和生物学，而且还提供了 为分支特异性抗生素开发的新目标提供平台，并指导我们如何增加 现有抗生素的有效性 上一个资助期的结果导致了分枝杆菌RNAP的高分辨率结构（通过 cryo-EM和晶体学），并提供了机会来表征Rif和Rif衍生物， 抑制Rif抗性（RifR）细菌抑制分枝杆菌RNAP。在这里，我们建议继续这一研究路线， 与由S.布雷迪，其抑制额外的RifR Mtb RNAP。 艰难梭菌（Clostrioides difficile，Cdiff）是一种革兰氏阳性、产芽孢、厌氧菌，是一种条件致病菌 这对受损宿主来说是致命的Fidaxomicin（Fdx）是FDA批准的唯一一种靶向 RNAP是治疗Cdiff感染的有效方法。我们最近的工作证实Fdx可以抑制Mtb RNAP， 但这种效力依赖于放线菌特异性转录因子RpbA， 不在Cdiff中。在这里，我们建议扩大我们的专业知识，在生化和结构研究的细菌 RNAP包括Cdiff所属的厚壁菌门的先前未表征的进化枝。此处的结果 将阐明Fdx效力的结构和生化基础，并提供结构和 生物化学的基础上开发Cdiff RNAP药物开发和优化。

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.