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

结核分枝杆菌和艰难梭菌 RNA 聚合酶及其调节剂的结构和功能表征

基本信息

批准号：
10586042
负责人：
ELIZABETH A CAMPBELL
金额：
$ 33.9万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2024-05-31
项目状态：
已结题

项目摘要

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.

项目概要由结核分枝杆菌 (Mtb) 引起的结核病 (TB) 仍然是一个重大的健康问题。美国疾病控制中心估计，世界上大约 1/3 至 1/4 的人口处于潜伏状态。已感染。 RNA 聚合酶 (RNAP) 是细菌中负责所有转录的酶，是利福霉素 (Rif) 类抗生素，结核病的一线治疗方法。因此 RNAP 是一种经过验证且新药开发的有吸引力的目标。这凸显了我们最近的结构性和 Mtb RNAP 的功能特征以及完整所需的两个必需转录因子的作用转录活性。之前的资助使我们能够提供 2.8 Å 分辨率的晶体结构来自耻垢分枝杆菌的 RNAP 转录起始复合物 (TIC) 以及最近 Mtb 的冷冻电镜结构转录复合物。在本提案中，冷冻电镜将用于检查 RNAP 复合物作为起点阐明相对未表征的转录因子家族 WhiB 因子的机制。 WhiB 因子仅存在于放线菌中，并且在 Mtb 中发挥作用，包括生长和生长的必要性。分裂以及对宿主诱导的应激的反应，包括抗生素耐受性、一氧化氮、巨噬细胞入侵和活性氧。我们将使用多学科方法，包括结构、生物化学、基因组和体内实验（与 J. Rock 合作）以了解其作用和这个重要但相对未知的转录因子家族的机制。结果来自这里的目标不仅有可能阐明这些因素的机制和生物学，而且还提供一个针对进化枝特异性抗生素开发新目标的平台，并指导我们如何增加目前抗生素的功效。上一资助期的结果已导致分枝杆菌 RNAP 的高分辨率结构（由冷冻电镜和晶体学），并提供了描述 Rif 和 Rif 衍生物如何抑制 Rif 抗性 (RifR) 细菌抑制分枝杆菌 RNAP。在此我们建议继续这一研究方向具有结构未表征的 Rif 衍生物，由 S. Brady 提供，可抑制额外的 RifR Mtb RNAP。艰难梭菌 (Cdiff) 是一种革兰氏阳性、产孢子、厌氧细菌，是一种机会性病原体这对于受感染的主机来说是致命的。 Fidaxomicin (Fdx)，FDA 批准的唯一一种靶向抗生素 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 有效，但这种效力取决于放线菌特异性转录因子 RpbA，它是 Cdiff 中不存在。在这里，我们建议扩展我们在细菌生化和结构研究方面的专业知识 RNAP 包括 Cdiff 所属的先前未表征的厚壁菌门分支。结果在这里将阐明 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。