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

项目摘要/摘要 驱动细菌细胞分裂的过程应该是新的设计的主要目标 抗生素，但我们对一种细菌如何变成两种细菌的理解存在着显著的差距 防止其被剥削。几乎所有的细菌都有相同的五蛋白复合体(FtsQLB-WI)来协调 在分裂细胞之间合成肽多聚(PG)隔膜。我们的实验室有丰富的经验 通过纯化和修饰脂质结合的PG前体来重建PG合成 在体外，我们对这种特定的PG合成复合体有深入的了解，因为我们是第一个 FtsW是一种PG糖基转移酶。因此，我们准备通过以下方式解决这一机制： 在分裂过程中，哪些病原体如肺炎链球菌调节PG的合成。在目标1中，我 将产生与PG前体结合的合成酶亚复合体FtsW和FTSI的结构 底物。FtsW是SEDS家族的一个新成员，这种结构将第一次定义FtsW如何 结合PG前体并提出一种作用机制。我们已经重组了PG合成酶 肺炎链球菌FtsWI的体外活性，发现加入FtsQLB对其有抑制作用。我们假设 蛋白质-蛋白质和底物的相互作用是FtsQLB-WI复合体降阻所必需的。在……里面 目的2，我将探索调节FtsWI合成酶活性的蛋白质因子，包括系统地鉴定 非酶亚复合体FtsQLb中调节FtsWI所需的结构域以及体内的结构域 免疫共沉淀法检测FtsQLB-WI以确定FtsQLB-WI所需的额外成分 去压抑。在目标3中，我将分离预测与调控结合的肽聚糖细胞壁的成分 FtsQLB-WI中的结构域，以确定它们对FtsQLB-WI体外活性的影响。加在一起，这些 AIMS将在肺炎链球菌中生成一个全面的间隔PG合成模型，为设计提供参考 新型抗生素。 这里提议的工作不仅将使我获得成为一名 独立研究人员，但另外也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，我将有足够的机会从事教学和 为高等教育事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，我将发展我的技能 要求成为本科院校的一名全面的微生物学研究人员和教育工作者。

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.