Phosphorylation and acetylation of secreted bacterial proteins: a new regulatory

分泌细菌蛋白的磷酸化和乙酰化：新的调控

• 批准号: 8778792
• 负责人: Cara Cheney Boutte
• 金额: $ 5.51万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8778792
• 关键词: Acetylation; Affect; Alleles; Antibiotic Resistance; Antibiotics; Antimycobacterial Agents; Antitubercular Agents; Bacteria; Bacterial Proteins; Biochemical; Cell Cycle; Cell Survival; Cell Wall; Cells; Cessation of life; Chemicals; Chromosome Segregation; Co-Immunoprecipitations; Cytolysis; Cytoplasm; Defect; Development; Drug Design; Drug Targeting; Elongation Factor; Enzymes; Event; Exhibits; Extracellular Protein; Fibrinogen; Genetic; Genus Mycobacterium; Goals; Growth; Homeostasis; Immune response; In Vitro; Infection; Length; Measures; Mediating; Modeling; Modification; Molecular; Mycobacterium tuberculosis; Nutrient; Peptidoglycan; Periplasmic Proteins; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Process; Proteins; Regulation; Research; Role; Signal Transduction; Stress; Structure; Techniques; Time; Work; cell growth; drug candidate; enzyme structure; extracellular; improved; mutant; mycobacterial; novel; overexpression; pathogen; periplasm; public health relevance; research study; scaffold; uptake

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how the cell wall of M. tuberculosis (Mtb) is regulated during cell growth and dormancy, and to identify drug candidates that will target cell wall enzymes in both of these conditions, thus making effective antibiotics. The objective of the research described in this proposal is to characterize, in Mtb and a related species, a previously-undescribed regulatory mechanism for secreted bacterial proteins. Work to date has shown that an essential cell elongation factor, CwlM, is phosphorylated and that this phosphorylation alters its activity in regulating cell elongation. The proposed work will probe the cellular and molecular function of CwlM by analyzing peptidoglycan structure and the localization of other elongation factors when CwlM is depleted, and will identify the regulatory targets of CwlM by using co-immunoprecipitation to identify protein interactors. A phosphoablative allele of CwlM that impairs cell elongation has been identified. The proposed work will measure the protein stability, and secretion of this mutant protein, and will analyze peptidoglycan structure and elongation factor localization in the mutant strain in order to understand how phosphorylation affects the activity of CwlM. In addition, the cognate kinase for CwlM will be identified. In an effort to understand how stress changes cell wall structure and growth dynamics, this work will study how stressful conditions alter the phosphorylation and activity of CwlM. This work will further the understanding of how secreted proteins - and especially those that remodel the cell wall - are regulated and how their activity is coordinated with cytoplasmic events. Understanding these processes is critical to building a model for how the cell wall is remodeled during both vegetative growth and the stress that leads to dormancy, and for identifying cell wall drug targets.

描述（由申请人提供）：这项工作的长期目标是了解M。结核病（Mtb）在细胞生长和休眠期间受到调节，并鉴定在这两种情况下靶向细胞壁酶的候选药物，从而制备有效的抗生素。本提案中描述的研究的目的是表征结核分枝杆菌和相关物种中分泌细菌蛋白的先前未描述的调节机制。迄今为止的工作已经表明，一种必需的细胞延伸因子CwlM被磷酸化，并且这种磷酸化改变了其在调节细胞延伸中的活性。本研究将通过分析CwlM缺失时的肽聚糖结构和其他延伸因子的定位来探讨CwlM的细胞和分子功能，并通过免疫共沉淀来鉴定蛋白质相互作用物来鉴定CwlM的调控靶点。已经鉴定了损害细胞伸长的CwlM的磷酸消融等位基因。拟议的工作将测量蛋白质的稳定性和这种突变蛋白的分泌，并将分析突变株中的肽聚糖结构和延伸因子定位，以了解磷酸化如何影响CwlM的活性。此外，将鉴定CwlM的同源激酶。为了了解压力如何改变细胞壁结构和生长动力学，这项工作将研究压力条件如何改变CwlM的磷酸化和活性。 这项工作将进一步了解分泌的蛋白质-特别是那些重塑细胞壁的蛋白质-是如何调节的，以及它们的活性如何与细胞质事件协调。了解这些过程对于建立一个模型来研究细胞壁在营养生长和导致休眠的应激过程中是如何重塑的，以及确定细胞壁药物靶点至关重要。