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.

项目总结/摘要 驱动细菌细胞分裂的过程应该是设计新型药物的主要目标。 抗生素，但我们对一种细菌如何变成两种细菌的理解存在重大差距， 防止其被剥削。几乎所有的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 通过纯化和修饰脂质结合的PG前体， 体外，而且，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表征为PG糖基转移酶。因此，我们完全有能力解决这一机制， 其中病原体如肺炎链球菌在分裂期间调节PG的合成。在目标1中， 将产生与PG前体结合的合酶亚复合物FtsW和FtsI的结构 衬底FtsW是SEDS家族的一个新成员，这个结构将是第一个定义FtsW如何 结合PG前体，并提出了一种作用机制。我们已经重建了PG合成酶 S.的活动。pneumoniae FtsWI体外培养，发现FtsQLB的加入具有抑制作用。我们假设 FtsQLB-WI复合物的去阻遏需要蛋白质-蛋白质和底物相互作用。在 目的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，其是FtsWI调节以及体内 针对FtsQLB-WI的免疫共沉淀试验，以鉴定FtsQLB-WI所需的其他组分 去压抑在目标3中，我将分离肽聚糖细胞壁的组分，预测其结合调节蛋白。 结构域内的FtsQLB-WI，以确定其对FtsQLB-WI在体外的活性的影响。所有这些 目的将产生一个全面的模式，间隔PG合成在S。肺炎，可以告知设计 新型抗生素。 这里提出的工作不仅会给我提供生物化学方面的专业知识， 独立的研究人员，但另外让我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，我将有充分的机会教书， 指导经验，为高等教育职业生涯做准备。我还将促进合作 在我的HMS社区以及更广泛的微生物学和本科生的专业关系 教育家社区作为HMS博士Suzanne步行者的博士后研究员，我将发展我的技能， 要求是一个全面的微生物学研究人员和教育工作者在本科院校。