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: 10226898
• 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/10226898
• 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; 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）细胞壁是一个巨大的网状分子，它决定细菌尺寸，形状和 链接，需要在宿主和环境领域中生存。克 - （+）细菌，如链球菌 肺炎，PG也充当其他表面大分子共价附着的支架。这 PG合成的调节是一个根本重要的空间和时间生物学问题，涉及 大量蛋白质合奏的相互作用，组装和拆卸以及这些蛋白质的表达 在压力期间正常生长和变化的水平。这笔赠款的长期目标是 确定调节细菌病原体PG合成的蛋白质相互作用和电路。 肺炎（肺炎球菌），用作这些机械，碱性的卵形细菌的模型 科学研究。该赠款将回答以下重要的，相互关联的有关肺炎球菌的问题 隔膜和周围（侧壁样）PG合成，它们均来自Midcell FTSZ环。从 FTSZ环，新的FTSZ环如何在新子细胞的赤道上找到和组装？什么是 蛋白质相互作用的方向运动和年代学，这些蛋白质组装和稳定FTSZ环在 细胞分裂的不同阶段？哪些角色是已知和新发现的调节蛋白及其角色 在FTSZ环组装和稳定和PG合成中，通过Ser/Thr激酶发挥的Ser/Thr激酶的磷酸化？移动 对于PG综合，携带机器的组成，方向运动和协调是什么 在细胞周期期间熄灭中隔和周围合成？哪些与调节蛋白的相互作用介导 B类青霉素结合蛋白（PBP）的单向运动沿成熟的隔环检测到？ A类PBP，SEDS经凝胶酶和调节的方式和相互作用是什么 在细胞周期期间平衡隔膜和周围PG合成的蛋白质？如何改变pg的突变 合成或其调节会影响PG组成和结构？关于PG重塑的相关主题，什么是 FTSEX激活PCSB PG水解酶活性的机制？哪种分裂蛋白与 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， 关于设定蛋白质量，KHPAB RNA结合蛋白在转录后如何调节 FTSA量，保守的KHPAB是否充当一般的RNA伴侣？第二使者如何 循环-DI-AMP调节肺炎球菌PG合成？代谢物前体途径的改变如何 对于PG综合，抑制了必需PBP的要求？这些问题将由 结合强大遗传，生理，细胞生物学的系统方法（例如，高分辨率3D-SIM 和TIRFM-SIM）和生化（例如UHPLC-MS/MS）方法来攻击此多组分问题。这 赠款将填补有关模型卵形形细菌中PG合成调节的主要空白，确定 报告的毒力因子的功能，并为抗生素发育提供了新的靶标和脆弱性。