Mechanistic Dissection and Antibiotic Discovery Targeting Clostridioides difficile RNA Polymerase

针对艰难梭菌 RNA 聚合酶的机制解析和抗生素发现

基本信息

批准号：
10523156
负责人：
Xinyun Cao
金额：
$ 10.45万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-12 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10523156
关键词：
Advisory Committees Alkaline Phosphatase Antibiotic Resistance Antibiotic Therapy Antibiotics Area Award Bacterial RNA Behavior Binding Biochemical Biochemistry Bioinformatics Biological Assay Biophysics Broccoli - dietary Centers for Disease Control and Prevention (U.S.)ChIP-seq Clostridium difficile Collaborations Communication Complex Cryoelectron Microscopy DNA DNA-Directed RNA Polymerase Data Set Development Dissection Docking Drug Targeting Educational process of instructing Elongation Factor Environmental Risk Factor Enzymes Escherichia coli Exhibits Flagella Future Gene Expression Gene Expression Profile Gene Expression Profiling Generations Genetic Genetic Transcription Genomics Goals Growth Healthcare Systems Human In Vitro Incidence Infection Investigation Kinetics Knowledge Leadership Life Mentors Molecular Molecular Conformation Multi-Drug Resistance Mus N-terminal Operon Organism Phase Physiology Play Prions Promoter Regions Protein Subunits Proteins Public Health RNA RNA Polymerase I RNA Polymerase Inhibitor Recombinants Recurrence Regulation Reporter Research Research Training Resistance Resources Ribosomal RNA Role Specificity Structure System Techniques Testing Texas Transcription Initiation Transcription Process Transcriptional Regulation United States Universities Virulence Wisconsin Work antimicrobial aptamer bacterial resistance base career combat design experimental study follow-up genetic information genome-wide high throughput screening host microbiome improved in vivo infectious disease treatment inhibitor insight mutant new therapeutic target non-prion novel paralogous gene pathogen pathogenic bacteria prion-like promoter response rho skills termination factor transcription factor transcription termination virtual screening

项目摘要

PROJECT SUMMARY With the alarming increase in the incidence of infections caused by antibiotic-resistance bacteria, there is an urgent need to identify new strategies to combat this emerging threat. The development, growth, and survival of all living organisms rely on coordinated gene expression. Central to gene expression is RNA polymerase (RNAP), a multi-subunit protein that transcribes genetic information from DNA to RNA in the complex and highly regulated process of transcription. Transcription has three major stages for creating a nascent RNA: initiation, elongation, and termination, each of which is controlled by protein transcription factors. RNAP is a proven drug target, but RNAP’s mechanistic features and how it is regulated by transcription factors remain poorly understood in pathogenic bacteria. My long-term goal is to understand the mechanisms of action of RNAP and key transcription factors involved in regulating RNAP initiation (CarD), elongation (NusG and NusA) and termination (Rho) in order to improve future antimicrobial development. In this proposed research, I will investigate the biochemical, structural, and genetic basis of the transcriptional machinery of Clostridioides difficile (C. diff.), a life-threatening gut pathogen that is resistant to multiple antibiotics. In Aim 1(K99 phase), I will investigate the functional relationship between two paralogs of the transcription regulator CarD and RNAP through in vitro and in vivo studies to test the hypothesis that the two CarD paralogs compete to bind and regulate RNAP, and the interplay of these factors is critical for coordinated control of transcription initiation in C. diff. In Aim 2 (K99/R00 phase), I will use genomic-scale mapping techniques and genetic assays to interrogate how Rho rewires gene expression by terminating transcription by RNAP. I will also design biochemistry assays to elucidate the mechanisms by which NusA and NusG, two universal elongation factors, modulate Rho-RNAP behavior. In Aim 3 (R00 phase), I will build an in vitro platform using the Broccoli fluorescent RNA aptamer to enable high-throughput screening of inhibitors of C. diff. RNAP. Virtual screening will be conducted to identify novel inhibitors based on our newly obtained cryo-EM structure. The proposed research in the K99 phase will mainly be conducted in the lab of Prof. Robert Landick at the University of Wisconsin-Madison. The key area that I will acquire additional research training is genome-scale mapping techniques and corresponding bioinformatics skills to analyze high-throughput datasets. I will also be guided by an advisory committee including collaborators Prof. Federico Rey (UW-Madison, an expert in microbiome-host interactions) and Prof. Elizabeth Campbell (The Rockefeller Univ., an expert in cryo-EM of RNAP and associated proteins), and consultant Prof. Joseph Sorg (Texas A&M Univ., an expert in C. diff genetics and physiology). I will also benefit from the facilities and abundant resources at UW-Madison. During the mentored phase of this award, I also plan to hone my skills in teaching, leadership and scientific communication, which will facilitate my transition to an independent research career.

项目概要随着抗生素耐药性细菌引起的感染发生率的惊人增加，迫切需要确定新的战略来应对这一新出现的威胁的发展、增长和生存。所有生物体都依赖于协调的基因表达基因表达的核心是RNA聚合酶。 (RNAP)，一种多亚基蛋白质，可将遗传信息从 DNA 转录为复杂的 RNA 转录过程受到高度调控，产生新生 RNA 分为三个主要阶段：起始、延伸和终止，每个过程都由蛋白质转录因子控制。已被证实的药物靶点，但 RNAP 的机制特征及其受转录因子调控的方式仍然存在我对致病细菌知之甚少，我的长期目标是了解其作用机制。 RNAP 和参与调节 RNAP 起始 (CarD)、延伸 (NusG 和 NusA) 的关键转录因子和终止（Rho），以改善未来的抗菌药物开发，在这项拟议的研究中，我将。研究梭菌转录机制的生化、结构和遗传基础艰难梭菌 (C. diff.)，一种对多种抗生素具有耐药性的危及生命的肠道病原体。将研究转录调节因子 CarD 和 RNAP 的两个旁系同源物之间的功能关系通过体外和体内研究来检验两个 CarD 旁系同源物竞争结合和调节RNAP，这些因素的相互作用对于转录起始的协调控制至关重要在目标 2（K99/R00 阶段）中，我将使用基因组规模作图技术和遗传分析来我还将设计探究 Rho 如何通过 RNAP 终止转录来重新连接基因表达。生物化学测定阐明了 NusA 和 NusG（两种通用伸长因子）的机制在 Aim 3（R00 阶段）中，我将使用 Broccoli 构建一个体外平台。荧光 RNA 适体可实现 C. diff 抑制剂的高通量筛选。将根据我们新获得的冷冻电镜结构来识别新型抑制剂。 K99阶段的研究将主要在英国大学Robert Landick教授的实验室进行我将获得额外研究培训的关键领域是基因组规模绘图。我还将指导分析高通量数据集的技术和相应的生物信息学技能。包括合作者 Federico Rey 教授（威斯康星大学麦迪逊分校，微生物组宿主专家）在内的咨询委员会相互作用）和Elizabeth Campbell教授（洛克菲勒大学，RNAP和冷冻电镜专家）相关蛋白）和顾问 Joseph Sorg 教授（德克萨斯农工大学，C. diff 遗传学和在指导期间，我还将受益于威斯康辛大学麦迪逊分校的设施和丰富的资源。在这个奖项的阶段，我还计划磨练我在教学、领导和科学沟通方面的技能，这将促进我向独立研究生涯的过渡。