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: 10655457
• 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/10655457
• 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; 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; Set protein; Shapes; Streptococcus pneumoniae; Stress; Structure; Superbug; Surface; System; Virulence Factors; biological adaptation to stress; daughter cell; genetic regulatory protein; macromolecule; pathogenic bacteria; posttranscriptional; 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）细胞壁是一个巨大的网状分子，决定了细菌的大小，形状， 链接，需要在主机和环境中生存的小生境。在革兰氏（+）细菌中，如链球菌 在肺炎链球菌中，PG还充当用于共价连接其它表面大分子的支架。的 PG合成的调节是一个基本上重要的空间和时间生物学问题， 相互作用，组装和拆卸的蛋白质和这些蛋白质的表达， 正常生长所需的水平，并在压力下发生变化。这项补助金的长期目标是 确定调节细菌病原体S中PG合成的蛋白质相互作用和回路。 肺炎（肺炎球菌），这是作为一个模型，卵形细菌在这些机制，基本- 科学研究。这项资助将回答以下关于肺炎球菌的重要的、相互关联的问题 隔膜和周边（侧壁样）PG合成，这两者都来自于中细胞FtsZ环。首先是 FtsZ环，新的FtsZ环如何在新的子细胞的赤道上找到并组装？有哪些 组装和稳定FtsZ环的蛋白质的相互作用的方向运动和时序， 细胞分裂的不同阶段已知和新发现的调节蛋白及其 丝氨酸/苏氨酸激酶磷酸化在FtsZ环组装和稳定以及PG合成中的作用？移动 到PG合成，携带机器的组成、方向运动和协调是什么？ 在细胞周期中的外隔膜和外周合成？与调节蛋白的相互作用介导 B类青霉素结合蛋白（PBPs）的单向运动检测沿着成熟隔环？ A类PBP、SEDS转糖基酶和调节性糖基转移酶的模式和相互作用是什么？ 在细胞周期中平衡间隔和外周PG合成的蛋白质？改变PG的突变是如何 合成或其调节影响PG组成和结构？关于PG重塑的相关话题， FtsEX激活PcsB PG水解酶活性的机制？哪些分裂体蛋白与 FtsEX：PcsB激活PG水解？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结合蛋白如何转录后调节 FtsA的量，保守的KhpAB作为一个一般的RNA伴侣？第二信使是如何 cyclic-di-AMP调节肺炎球菌PG合成？代谢物前体途径的改变 抑制对必需PBPs的需求？这些问题将由一个 系统方法结合了强大的遗传学，生理学，细胞生物学（例如，高分辨率三维SIM 和TIRFm-SIM），和生物化学（例如，UHPLC-MS/MS）方法来解决该多组分问题。这 格兰特将填补关于在一个模型卵形细菌中PG合成调控的主要空白， 研究了已报道的毒力因子的功能，并为抗生素的开发提供了新的靶点和弱点。

项目成果

S1 Domain RNA-Binding Protein CvfD Is a New Posttranscriptional Regulator That Mediates Cold Sensitivity, Phosphate Transport, and Virulence in Streptococcus pneumoniae D39.

S1 结构域 RNA 结合蛋白 CvfD 是一种新型转录后调节因子，可介导肺炎链球菌 D39 的冷敏感性、磷酸盐转运和毒力。

• DOI: 10.1128/jb.00245-20
• 发表时间: 2020
• 期刊: Journal of bacteriology
• 影响因子: 3.2
• 作者: Sinha,Dhriti;Zheng,JiaqiJ;Tsui,Ho-ChingTiffany;Richardson,JohnD;DeLay,NicholasR;Winkler,MalcolmE
• 通讯作者: Winkler,MalcolmE

The Pneumococcal Divisome: Dynamic Control of Streptococcus pneumoniae Cell Division.

• DOI: 10.3389/fmicb.2021.737396
• 发表时间: 2021
• 期刊: Frontiers in microbiology
• 影响因子: 5.2
• 作者: Briggs NS;Bruce KE;Naskar S;Winkler ME;Roper DI
• 通讯作者: Roper DI

Undermodification cues division.

• DOI: 10.1038/s41589-021-00818-2
• 发表时间: 2021-08
• 期刊: NATURE CHEMICAL BIOLOGY
• 影响因子: 14.8
• 作者: Winkler, Malcolm E.