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

调节超级细菌肺炎链球菌肽聚糖合成的动力学、稳态和应激反应的新调控相互作用和回路

• 批准号: 10452519
• 负责人: MALCOLM E. WINKLER
• 金额: $ 65.5万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452519
• 关键词: 3-Dimensional; Affect; Antibiotic Resistance; Antibiotics; Bacteria; Basic Science; Biochemical; Biological; Cell Cycle; Cell Separation; Cell Wall; Cell division; Cells; Chronology; Development; Equilibrium; Future; Genetic; Genetic Transcription; Goals; Grant; Growth; Health; Homeostasis; Human; Hydrolysis; Mediating; Methods; Modality; Modeling; Molecular Chaperones; Movement; Mutation; N-Acetylmuramoyl-L-alanine Amidase; Pathway interactions; Penicillin-Binding Proteins; Peptidoglycan; Periodicity; Peripheral; Phosphorylation; Phosphotransferases; Physiological; Play; Proteins; RNA; RNA-Binding Proteins; Regulation; Reporting; Resolution; Role; Second Messenger Systems; Shapes; Streptococcus pneumoniae; Stress; Structure; Superbug; Surface; System; Virulence Factors; biological adaptation to stress; daughter cell; genetic regulatory protein; macromolecule; pathogenic bacteria; protein expression; scaffold

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.

肽聚糖 (PG) 细胞壁是一种巨大的网状分子，决定细菌的大小、形状和结构。 链，是在宿主和环境生态位中生存所必需的。在链球菌等革兰氏 (+) 细菌中 在肺炎链球菌中，PG 还充当其他表面大分子共价附着的支架。这 PG合成的调控是一个极其重要的时空生物学问题，涉及 大量蛋白质的相互作用、组装和分解以及这些蛋白质的表达 适合正常生长的水平，但在压力期间会发生变化。这笔赠款的长期目标是 确定细菌病原体 S 中调节 PG 合成的蛋白质相互作用和电路。 肺炎球菌（肺炎球菌），它被用作这些机械的、基本的卵形细菌的模型 科学研究。这笔赠款将回答以下有关肺炎球菌的重要且相互关联的问题 隔膜和外周（侧壁样）PG 合成，均源自细胞中 FtsZ 环。开始于 FtsZ 环，新的 FtsZ 环如何在新子细胞的赤道处找到并组装？有哪些 组装并稳定 FtsZ 环的蛋白质相互作用的定向运动和时间顺序 细胞分裂的不同阶段？已知和新发现的调节蛋白及其作用有何作用 Ser/Thr 激酶的磷酸化在 FtsZ 环组装和稳定以及 PG 合成中发挥作用？移动 PG合成，承载的机器的组成、定向运动、协调性是怎样的 细胞周期中的隔膜和外周合成？与调节蛋白的哪些相互作用介导 B 类青霉素结合蛋白 (PBP) 沿着成熟间隔环检测到的单向运动？ A 类 PBP、SEDS 转糖基酶和监管的模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？突变如何改变 PG 合成或其调控影响PG的组成和结构？关于PG重构的相关话题，什么是 FtsEX 激活 PcsB PG 水解酶活性的机制是什么？哪些分裂体蛋白质相互作用 FtsEX：PcsB 可以激活 PG 水解吗？ FtsEX:PcsB 在细胞分离中的主要作用是什么？最后， 关于设定蛋白质含量，KhpAB RNA 结合蛋白如何进行转录后调节 FtsA 量，保守的 KhpAB 是否充当一般 RNA 伴侣？第二使者如何 环二AMP调节肺炎球菌PG合成？代谢物前体途径的改变如何影响 PG 合成是否会抑制必需 PBP 的需求？这些问题将由 结合了强大的遗传、生理、细胞生物学功能的系统方法（例如高分辨率 3D-SIM 和 TIRFm-SIM）和生物化学（例如 UHPLC-MS/MS）方法来解决这个多组分问题。这 拨款将填补关于卵形细菌模型中 PG 合成调节的主要空白，确定 已报道的毒力因子的功能，并为抗生素的开发提供新的目标和脆弱性。