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

• 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
• 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

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.

项目摘要 随着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。发展、成长和生存 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转录为复合体中的RNA， 高度调节的转录过程。转录有三个主要阶段来产生新生RNA： 起始、延伸和终止，其中每一个都由蛋白质转录因子控制。RNAP是一个 RNAP是一个已被证实的药物靶点，但RNAP的机制特征以及它如何受转录因子的调控仍然存在 对病原菌知之甚少。我的长期目标是了解 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），以改善未来的抗微生物开发。在这项研究中，我将 研究梭菌属转录机制的生化、结构和遗传基础 艰难梭菌（C. diff.），一种对多种抗生素有抗药性的致命肠道病原体。在目标1（K99阶段）中， 将研究转录调节因子CardD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种CardD旁系同源物竞争结合和 调节RNAP，这些因素的相互作用对于协调控制转录起始至关重要。 C. diff.在Aim 2（K99/R 00阶段），我将使用基因组规模作图技术和遗传分析， 询问Rho如何通过RNAP终止转录来重新连接基因表达。我也会设计 生物化学测定来阐明NusA和NusG，两种通用延伸因子， 调节Rho-RNAP行为。在目标3（R 00阶段），我将使用花椰菜构建一个体外平台 荧光RNA适体，以实现高通量筛选C. diff. RNAP。虚拟筛选 将进行鉴定新的抑制剂的基础上，我们新获得的冷冻EM结构。拟议 K99阶段的研究将主要在密歇根大学罗伯特·兰迪克教授的实验室进行。 威斯康星州麦迪逊。我将获得额外的研究培训的关键领域是基因组规模的映射 技术和相应的生物信息学技能来分析高通量数据集。我也将遵循 一个咨询委员会，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） 相互作用）和伊丽莎白坎贝尔教授（洛克菲勒大学，RNAP冷冻电镜专家， 相关蛋白）和顾问Joseph索尔格教授（德克萨斯A&M大学，C的专家。不同的基因和 生理学）。我也将受益于威斯康星大学麦迪逊分校的设施和丰富的资源。在指导期间， 在这个奖项的第一阶段，我还计划磨练我在教学，领导和科学交流方面的技能， 将帮助我过渡到独立的研究生涯。