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.

项目摘要/摘要 驱动细菌细胞分裂的过程应该是设计新颖的主要目标 抗生素，但是我们对一个细菌如何变成两个细菌的理解充斥着明显的差距 防止其剥削。几乎所有细菌都具有相同的五蛋白复合物（FTSQLB-WI）以坐标 分裂细胞之间肽聚糖（PG）隔膜的合成。我们的实验室有丰富的经验 随着脂质结合的PG前体的纯化和修饰，以重构PG合成 体外，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的特征视为PG糖基转移酶。因此，我们有独特的准备解决机制 哪些病原体（例如肺炎链球菌）调节分裂过程中PG的合成。在AIM 1中，我 将生成与PG前体结合的合成酶子复合物，FTSW和FTSI的结构 基材。 FTSW是SEDS家庭的新成员，该结构将是第一个定义FTSW的结构 结合PG前体并提出一种作用机理。我们已经重构了PG合酶 肺炎链球菌在体外的活性，发现添加FTSQLB是抑制性的。我们假设这一点 蛋白质 - 蛋白质和底物相互作用都是FTSQLB-WI复合物的抑制所必需的。在 AIM 2，我将探索调节FTSWI合酶活性的蛋白质因子，包括对 FTSWI调节所需的非酶子子复合ftsqlb中的域以及体内 针对FTSQLB-WI的共免疫沉淀测定法，以识别FTSQLB-WI所需的其他组件 消除抑制。在AIM 3中，我将隔离肽聚糖细胞壁的组件，预计会结合调节 FTSQLB-WI中的域，以确定它们对体外FTSQLB-WI活性的影响。在一起，这些 AIMS将在肺炎链球菌中产生一个综合的分子PG合成模型，可以告知设计 新型抗生素。 这里提出的工作不仅会授予我成为生物化学方面的专业知识 独立的研究人员，但此外，我还可以接近其他重要的专业手段 发展。在哈佛医学院和波士顿地区，我将有足够的教学机会 指导为高等教育事业做准备的经验。我还将培养合作 我的HMS社区以及更广泛的微生物学和本科生的专业关系 教育社区。作为HMS Suzanne Walker博士领导的博士后研究员，我将发展我的技能 要求成为本科机构的全面微生物研究人员和教育者。