New Regulatory Interactions and Circuits that Mediate the Dynamics, Homeostasis, and Stress Responses of Peptidoglycan Synthesis in the Superbug Streptococcus pneumoniae

肽聚糖(PG)细胞壁是一个巨大的网状分子，它决定了细菌的大小、形状和 链，在宿主和环境生态位中生存所必需的。在革兰氏(G)细菌中，如链球菌 Pg还可作为其他表面大分子共价连接的支架。这个 PG合成的调控是一个基本的重要的时空生物学问题，涉及 大量蛋白质的相互作用、组装和拆解以及这些蛋白质在 正常生长所需的水平，并在压力下发生变化。这笔赠款的长期目标是 确定调节细菌病原体S. 肺炎球菌，作为卵球状细菌的模型，在这些机械的，基本的- 科学研究。这笔赠款将回答关于肺炎球菌的下列重要的、相互关联的问题 间隔和外周(侧壁样)PG合成，两者都来自中细胞FtsZ环。从开始 FtsZ环，新的FtsZ环是如何在新的子细胞的赤道上发现和组装的？什么是 组装并稳定FtsZ环的蛋白质相互作用的定向运动和年代学 细胞分裂的不同阶段？已知的和新发现的调节蛋白以及它们的 丝氨酸/苏氨酸激酶的磷酸化在FtsZ环组装和稳定以及PG合成中起作用？搬家 对于PG合成，机器的组成、定向运动和协调是什么 在细胞周期中隔膜和外周合成？与调节蛋白的哪些相互作用起中介作用 B类青霉素结合蛋白(PBPs)沿成熟隔环的单向运动？ A类PBPs、SEDS转糖基酶和调节性糖基酶的形式和相互作用是什么 在细胞周期中平衡隔膜和外周PG合成的蛋白质？基因突变是如何改变PG的 PG的合成或调节是否影响PG的组成和结构？关于PG重塑的相关话题，什么是 FtsEX激活PCSB PG水解酶活性的机制是什么？分裂体蛋白与哪些蛋白质相互作用 FtsEX：PCSB激活PG水解酶？FtsEX：PCSB在细胞分离中的主要作用是什么？最后， 关于设定蛋白量，KhpAB RNA结合蛋白是如何转录后调节的 FTSA含量，保守的KhpAB是否作为一般的RNA伴侣？第二个信使是如何 环二AMP调节肺炎链球菌PG合成？代谢物前体途径的改变是如何 PG的合成抑制了对必需的PBPs的需求？这些问题将由一个 结合了强大的遗传、生理、细胞生物学(例如高分辨率3D-SIM)的系统方法 和TIRFM-SIM)和生化方法(例如，UHPLC-MS/MS)来解决这一多组分问题。这 格兰特将填补关于卵形细菌模型中PG合成调控的主要空白，确定 已报告的毒力因子的功能，并为抗生素的开发提供新的靶点和脆弱性。

The peptidoglycan (PG) cell wall is a gigantic mesh-like molecule that determines bacterial size, shape, and chaining, required for survival in hosts and environmental niches. In Gram-(+) bacteria like Streptococcus pneumoniae, PG also acts as the scaffold for covalent attachment of other surface macromolecules. The regulation of PG synthesis is a fundamentally important spatial and temporal biological problem that involves interactions, assembly, and disassembly of a large ensemble of proteins and expression of these proteins at levels that are correct for normal growth and changed during stress. The long-term goal of this grant is to determine the protein interactions and circuits that regulate PG synthesis in the bacterial pathogen, S. pneumoniae (pneumococcus), which is used as a model for ovoid-shaped bacteria in these mechanistic, basic- science studies. This grant will answer the following important, interrelated questions about pneumococcal septal and peripheral (sidewall-like) PG synthesis, which both emanate from midcell FtsZ rings. Starting with FtsZ rings, how do new FtsZ rings find and assemble at equators of new daughter cells? What are the directional movements and chronology of interactions of proteins that assemble and stabilize the FtsZ ring at different stages of cell division? What roles do known and newly discovered regulatory proteins and their phosphorylation by a Ser/Thr kinase play in FtsZ ring assembly and stabilization and in PG synthesis? Moving to PG synthesis, what are the composition, directional movement, and coordination of the machines that carry out septal and peripheral synthesis during the cell cycle? Which interactions with regulatory proteins mediate the unidirectional movement of Class B penicillin-binding proteins (PBPs) detected along mature septal rings? What are the modalities and interactions of the Class A PBPs, SEDS transglycosylases, and regulatory proteins that balance septal and peripheral PG synthesis during the cell cycle? How do mutations that alter PG synthesis or its regulation affect PG composition and structure? On the related topic of PG remodeling, what is the mechanism by which FtsEX activates PcsB PG hydrolase activity? Which divisome proteins interact with FtsEX:PcsB to activate PG hydrolysis? What is the primary role of FtsEX:PcsB in cell separation? Finally, regarding setting protein amounts, how does the KhpAB RNA binding protein post-transcriptionally regulate FtsA amount, and does conserved KhpAB act as a general RNA chaperone? How does the second messenger cyclic-di-AMP regulate pneumococcal PG synthesis? How does alteration of the metabolite precursor pathway for PG synthesis suppress the requirement for essential PBPs? These questions will be answered by a systems approach that combines powerful genetic, physiological, cell biological (e.g., high-resolution 3D-SIM and TIRFm-SIM), and biochemical (e.g., UHPLC-MS/MS) methods to attack this multicomponent problem. This grant will fill in major gaps about the regulation of PG synthesis in a model ovoid-shaped bacterium, identify functions of reported virulence factors, and provide new targets and vulnerabilities for antibiotic development.