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.

项目摘要由结核分枝杆菌（MTB）引起的结核病（TB）继续构成重大健康问题。疾病控制中心估计，大约1/3至1/4的人口是潜在的已感染。 RNA聚合酶（RNAP），负责细菌中所有转录的酶是利福米霉素（RIF）类抗生素，一种针对结核病的第一线治疗。因此，RNAP是一个经过验证的开发新药的有吸引力的目标。这突出了我们最近结构的重要性 MTB RNAP的功能表征和完整所需的两个基本转录因子的作用转录活动。以前的赠款使我们能够提供2.8Å的分辨率晶体结构 M. smegmatis和MTB的Cryo-EM结构的RNAP转录启动复合物（TIC）转录复合物。在此提案中，Cryo-EM将用于检查RNAP复合物作为起点为了阐明一个相对未表征的转录因子的机制，即whib因子。 WHIB因素仅在肌细菌中发现，并且在MTB中具有作用，其中包括生长和分裂以及对宿主诱导应力的反应，包括抗生素耐受性，一氧化氮，巨噬细胞入侵和活性氧。我们将使用包括结构性的多学科方法生化，基因组和体内实验（与J. Rock合作），以了解角色和这种重要但相对未表征的转录因子家族的机制。来自此处的目的不仅有可能阐明这些因素的机制和生物学，而且还提供了一个新目标的平台，用于特异性抗生素开发，并为我们指导如何增加当前抗生素曲目的效率。上一个资金期的结果导致了分枝杆菌RNAP的高分辨率结构（通过冷冻EM和晶体学），并提供了表征RIF和RIF衍生物的机会抑制RIF抗性（RIFR）细菌抑制分枝杆菌RNAP。在这里，我们建议继续进行这一研究由S. Brady提供的结构未表征的RIF衍生物，抑制了其他RIFR MTB RNAP。梭状芽胞杆菌艰难梭菌（CDIFF），一种革兰氏阳性，孢子的，厌氧细菌，是一种机会性病原体这是致命的，这是妥协的主机。 Fidaxomicin（FDX），唯一的其他FDA批准的抗生素针对 RNAP是CDIFF感染的强大治疗方法。我们最近的工作确定FDX可以抑制MTB RNAP 可能的是，但这种效力取决于特异性转录因子RPBA，这是在CDIFF中不存在。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 rnaps包括CDIFF所属的先前未表征的公司。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。