Peptidoglycan Biogenesis in Escherichia Coli

大肠杆菌中的肽聚糖生物合成

• 批准号: 8602803
• 负责人: Thomas G Bernhardt
• 金额: $ 41.34万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8602803
• 关键词: ATP-Binding Cassette Transporters; Address; Alanine; Amidohydrolases; Anti-Bacterial Agents; Antibiotic Therapy; Antibiotics; Bacteria; Biochemical; Biogenesis; Biological; Biological Models; Cell Separation; Cell Shape; Cells; Co-Immunoprecipitations; Code; Coupled; Crutches; Cytokinesis; Cytolysis; Development; Enzymes; Equilibrium; Escherichia coli; Future; Genes; Genetic; Goals; Growth; Hydrolysis; In Vitro; Investigation; Lactams; Life; Lipoprotein (a); Lysostaphin; Lytic; Maintenance; Mediating; Membrane; Molecular; Monobactams; Mutation; N-Acetylmuramoyl-L-alanine Amidase; Pathway interactions; Penicillin-Binding Proteins; Penicillins; Peptidoglycan; Peptidyltransferase; Phase; Polymers; Polysaccharides; Process; Proteins; Reaction; Regulation; Reliance; Research Proposals; Role; Rupture; Shapes; Site; Surface; System; Vancomycin; Work; amidase; base; cell growth regulation; crosslink; daughter cell; follow-up; genetic analysis; in vivo; mutant; novel; operation; periplasm; prevent; prospective; protein function; protein protein interaction; public health relevance; research study; screening; yeast two hybrid system

DESCRIPTION (provided by applicant): Most bacteria surround themselves with a crosslinked polysaccharide polymer called peptidoglycan (PG) that is critical for the maintenance of cell shape and integrity. Because of its essentiality, surface exposure, and uniqueness to bacteria, the PG synthetic pathway has historically been an effective target for many of our most important antibacterial treatments like penicillin and vancomycin. Penicillin targets the PG synthases called the penicillin binding proteins (PBPs). These enzymes come in several varieties, but the major cellular PG synthases are thought to be the bi-functional PBPs because they possess both the transglycosylase and transpeptidase activities needed to synthesize the glycan strands of PG and crosslink them, respectively. Despite their prominence as antibiotic targets, we still do not understand how the bi-functional PBPs assemble the cell-shaped PG meshwork or what additional factors might help them accomplish this task. One of the principle reasons for this has been an over-reliance on penicillin and other antibiotics as probes for the identification of important PG assembly factors. To extend our experimental reach beyond the "crutch" of antibiotic probes, we developed a genetic approach to identify factors needed for proper PBP function in vivo using E. coli as a model system. E. coli encodes three bi-functional PBPs: PBP1A, PBP1B, and PBP1C. Each one is individually dispensable, but the simultaneous inactivation of both PBP1A and PBP1B leads to rapid cell lysis. Based on the essentiality of the PBP1A/PBP1B combination, we reasoned that factors required to promote PBP1A activity could be identified by screening for mutants synthetically lethal with the loss of PBP1B (slb mutants) and vice versa. Using this approach, we have implicated several known division proteins and a lipoprotein of unknown function in the assembly of PG by PBP1A. In the first two aims of this proposal we describe genetic, cell biological, and biochemical experiments intended to investigate the connection between PBP1A and these factors. These studies will help us determine whether or not the Slb factors are directly interacting with and/or influencing either of the two enzymatic activities of PBP1A. In related work, we discovered that the EnvC protein is likely to be an activator of the PG hydrolases (amidases) AmiA and AmiB that stimulates their activity to bring about daughter cell separation during cytokinesis. Specific Aim 3 seeks to determine how EnvC and the amidases cooperate to perform such a delicate operation without causing a lethal breach in the PG layer. We will begin addressing this by defining the mechanism by which EnvC might activate the amidases and identifying regulators of this activation activity. The long term goal of our work is to develop a molecular understanding of PG assembly by the PBPs and how it is remodeled in a controlled fashion by PG hydrolases. By gaining this understanding we hope to uncover new ways to disrupt the cellular balance between PG synthesis and hydrolysis for the development of novel classes of lytic antibiotics.

描述（由申请人提供）：大多数细菌用称为肽聚糖（PG）的交联多糖聚合物包围，这对于维持细胞形状和完整性至关重要。由于它的重要性，表面暴露和对细菌的独特性，PG合成途径历史上一直是我们许多最重要的抗菌治疗（如青霉素和万古霉素）的有效靶标。青霉素靶向称为青霉素结合蛋白（PBP）的PG合酶。这些酶有几种品种，但是主要的细胞PG合酶被认为是双功能PBP，因为它们既具有分别合成PG的聚糖链并交叉链接所需的经糖基酶和经肽酶活性。尽管它们是抗生素靶标的突出，但我们仍然不了解双功能PBP如何组装细胞形PG网格工作，或者哪些其他因素可能有助于他们完成这项任务。造成这种情况的主要原因之一是过度依赖青霉素和其他抗生素，作为识别重要PG组装因子的探针。为了将实验范围扩展到抗生素探针的“拐杖”之外，我们开发了一种遗传方法，以使用大肠杆菌作为模型系统来鉴定体内适当的PBP功能所需的因素。大肠杆菌编码三个双功能PBP：PBP1A，PBP1B和PBP1C。每个人都可以单独配置，但是PBP1A和PBP1B的同时失活导致了快速的细胞裂解。基于PBP1A/PBP1B组合的重要性，我们认为可以通过筛选突变体合成致死的PBP1B（SLB突变体）（SLB突变体）的损失，反之亦然。使用这种方法，我们牵涉到PBP1A组装中的几种已知分裂蛋白和未知功能的脂蛋白。在该提案的前两个目标中，我们描述了旨在研究PBP1A与这些因素之间的联系的遗传，细胞生物学和生化实验。这些研究将帮助我们确定SLB因子是否直接与PBP1A酶促活性中的任何一种直接相互作用和/或影响。在相关工作中，我们发现ENVC蛋白可能是PG水解酶（amidass）Amia和Amib的激活剂，它们刺激其活性，以在细胞分裂过程中引起子细胞分离。特定的目标3试图确定环境环境和胺化酶如何合作以进行如此微妙的操作，而不会在PG层中引起致命的破坏。我们将通过定义ENVC激活胺化酶并识别该激活活动的调节剂的机制来解决这一问题。我们工作的长期目标是通过PBPS对PG组装进行分子理解，以及如何通过PG水解酶以控制的方式重塑其。通过获得这种理解，我们希望发现新的方法来破坏PG合成与水解水解之间的细胞平衡，以开发新型的裂解抗生素类。