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）：大多数细菌周围都有一种称为肽聚糖（PG）的交联多糖聚合物，它对于维持细胞形状和完整性至关重要。由于其重要性、表面暴露性以及对细菌的独特性，PG 合成途径历来都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 合成酶。这些酶有多种，但主要的细胞 PG 合酶被认为是双功能 PBP，因为它们分别具有合成 PG 聚糖链和交联它们所需的转糖基酶和转肽酶活性。尽管它们作为抗生素靶标具有突出地位，但我们仍然不了解双功能 PBP 如何组装细胞形状的 PG 网络，或者还有哪些其他因素可以帮助它们完成这项任务。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鉴定重要 PG 组装因子的探针。为了将我们的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，使用大肠杆菌作为模型系统来识别体内 PBP 正常功能所需的因素。大肠杆菌编码三种双功能 PBP：PBP1A、PBP1B 和 PBP1C。每一种都是单独可有可无的，但 PBP1A 和 PBP1B 同时失活会导致细胞快速裂解。基于 PBP1A/PBP1B 组合的重要性，我们推断促进 PBP1A 活性所需的因子可以通过筛选 PBP1B 缺失的综合致死突变体（slb 突变体）来鉴定，反之亦然。使用这种方法，我们发现几种已知的分裂蛋白和一种功能未知的脂蛋白参与了 PBP1A 组装 PG 的过程。在本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 的两种酶活性相互作用和/或影响其中任何一种。在相关工作中，我们发现EnvC蛋白很可能是PG水解酶（酰胺酶）AmiA和AmiB的激活剂，刺激它们的活性，在胞质分裂过程中引起子细胞分离。具体目标 3 旨在确定 EnvC 和酰胺酶如何合作执行如此微妙的操作，而不会对 PG 层造成致命的破坏。我们将通过定义 EnvC 激活酰胺酶的机制并确定该激活活性的调节因子来开始解决这个问题。我们工作的长期目标是发展对 PBP 组装 PG 以及如何通过 PG 水解酶以受控方式重塑的分子理解。通过了解这一点，我们希望发现破坏 PG 合成和水解之间细胞平衡的新方法，以开发新型溶解性抗生素。