Biochemical and structural characterization of the cell wall synthesis complex required for bacterial division

细菌分裂所需的细胞壁合成复合物的生化和结构表征

• 批准号: 10750639
• 负责人: Anna I Weaver
• 金额: $ 6.91万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750639
• 关键词: Active Sites; Antibiotics; Area; Bacteria; Bacterial Infections; Binding; Biochemical; Biochemistry; Biological Assay; Boston; Cell Wall; Cell division; Cells; Cellular Structures; Cessation of life; Co-Immunoprecipitations; Communities; Complement; Complex; Crystallization; Data; Development; Drug Design; Drug Targeting; Drug resistance; Educational process of instructing; Enzyme Interaction; Enzymes; Family; Fostering; Genetic; Gram-Negative Bacteria; Gram-Positive Bacteria; Grant; In Vitro; Institution; Investigation; Knowledge; Laboratories; Ligands; Lipid Binding; Mediating; Membrane; Mentors; Microbiology; Modeling; Modification; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan; Peptidoglycan glycosyltransferase; Peptidyltransferase; Physiological; Polymers; Postdoctoral Fellow; Preparation; Process; Protein Family; Protein Region; Proteins; Regulation; Research Personnel; Streptococcus pneumoniae; Structure; Substrate Interaction; Teichoic Acids; Testing; Work; assay development; career; derepression; design; enzyme activity; experience; experimental study; glycosyltransferase; higher education; in vitro activity; in vivo; inhibitor; insight; medical schools; member; mutant; novel; novel antibiotic class; pathogen; polymerization; prevent; preventable death; protein complex; reconstitution; skills; undergraduate student

PROJECT SUMMARY/ABSTRACT The processes that drive bacterial cell division should be prime targets for the design of novel antibiotics, but our understanding of how one bacterium becomes two is riddled with significant gaps that prevent its exploitation. Nearly all bacteria possess the same five-protein complex (FtsQLB-WI) to coordinate the synthesis of a peptidoglycan (PG) septum between dividing cells. Our laboratory has extensive experience with the purification and modification of lipid-bound PG precursors for the reconstitution of PG synthesis in vitro, and, we have intimate knowledge of this specific PG synthesis complex as we were the first to characterize FtsW as a PG glycosyltransferase. We are therefore uniquely poised to resolve the mechanism by which pathogens such as Streptococcus pneumoniae regulate the synthesis of PG during division. In Aim 1, I will generate a structure of the synthase enzyme subcomplex, FtsW and FtsI, bound to PG precursor substrate. FtsW is a novel member of the SEDS-family, and this structure will be the first to define how FtsW binds PG precursors and suggest a mechanism of action. We have already reconstituted the PG synthase activity of S. pneumoniae FtsWI in vitro and found that addition of FtsQLB is inhibitory. We hypothesize that both protein-protein and substrate interactions are required for de-repression of the FtsQLB-WI complex. In Aim 2, I will explore protein factors that regulate FtsWI synthase activity, including a systematic identification of domains within the non-enzymatic subcomplex FtsQLB that are required for FtsWI regulation, as well as in vivo co-immunoprecipitation assays against FtsQLB-WI to identify additional components required for FtsQLB-WI de-repression. In Aim 3, I will isolate components of the peptidoglycan cell wall predicted to bind regulatory domains within FtsQLB-WI to determine their influence on the activity of FtsQLB-WI in vitro. Together, these aims will generate a comprehensive model of septal PG synthesis in S. pneumoniae that can inform the design of novel antibiotics. The work proposed here will not only grant me the expertise in biochemistry I require to become an independent researcher, but additionally place me in proximity to other important means of professional development. At Harvard Medical School and in the Boston area, I will have ample access to teaching and mentoring experiences in preparation for a career in higher education. I will also foster collaborative professional relationships within my HMS community as well as the broader microbiology and undergraduate educator community. As a postdoctoral fellow under Dr. Suzanne Walker at HMS, I will develop the skills I require to be a well-rounded microbiology researcher and educator at an undergraduate institution.

项目概要/摘要 驱动细菌细胞分裂的过程应该是设计新型细菌的主要目标 抗生素，但我们对一种细菌如何变成两种细菌的​​理解充满了巨大的差距 防止其被利用。几乎所有细菌都具有相同的五蛋白复合物（FtsQLB-WI）来协调 分裂细胞之间肽聚糖（PG）隔膜的合成。我们的实验室拥有丰富的经验 纯化和修饰脂质结合的 PG 前体，以重建 PG 合成 体外，并且，我们对这种特定的 PG 合成复合物有深入的了解，因为我们是第一个 将 FtsW 描述为 PG 糖基转移酶。因此，我们准备通过以下方式解决该机制： 肺炎链球菌等病原体在分裂过程中调节 PG 的合成。在目标 1 中，我 将生成与 PG 前体结合的合酶亚复合物 FtsW 和 FtsI 的结构 基材。 FtsW 是 SEDS 系列的新成员，该结构将第一个定义 FtsW 如何 结合 PG 前体并提出作用机制。我们已经重建了 PG 合酶 体外研究肺炎链球菌 FtsWI 的活性，发现添加 FtsQLB 具有抑制作用。我们假设 蛋白质-蛋白质和底物相互作用都是 FtsQLB-WI 复合物去抑制所必需的。在 目标 2，我将探索调节 FtsWI 合酶活性的蛋白质因子，包括系统鉴定 非酶亚复合物 FtsQLB 内的结构域是 FtsWI 调节以及体内所需的 针对 FtsQLB-WI 的免疫共沉淀分析，以确定 FtsQLB-WI 所需的其他成分 去压抑。在目标 3 中，我将分离预计能结合调节因子的肽聚糖细胞壁成分 FtsQLB-WI 中的结构域，以确定它们对 FtsQLB-WI 体外活性的影响。在一起，这些 目标将生成肺炎链球菌中间隔 PG 合成的综合模型，为设计提供信息 新型抗生素。 这里提出的工作不仅会给我提供成为一名生物化学专家所需的专业知识 独立研究员，但也让我接近其他重要的专业途径 发展。在哈佛医学院和波士顿地区，我将有充足的机会接触教学和 为高等教育职业做准备的指导经验。我还将促进协作 我的 HMS 社区内的专业关系以及更广泛的微生物学和本科生 教育者社区。作为 HMS Suzanne Walker 博士的博士后研究员，我将培养我所掌握的技能 要求成为本科院校的全面微生物学研究员和教育家。