Peptidoglycan Biogenesis in Escherichia Coli

性状（由申请方提供）：大多数细菌被称为肽聚糖（PG）的交联多糖聚合物包围，肽聚糖对维持细胞形状和完整性至关重要。由于其对细菌的重要性、表面暴露和独特性，PG合成途径历来是我们许多最重要的抗菌治疗（如青霉素和万古霉素）的有效靶点。青霉素靶向称为青霉素结合蛋白（PBP）的PG酶。这些酶有几种，但主要的细胞PG糖苷酶被认为是双功能的PBP，因为它们具有合成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的突出地位，我们仍然不了解双功能PBPs如何组装细胞状PG网络，或者还有什么其他因素可以帮助它们完成这项任务。其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E.大肠杆菌作为模型系统。E.大肠杆菌编码三种双功能PBPs：PBP 1A、PBP 1B和PBP 1C。每一个都是单独灭活的，但PBP 1A和PBP 1B同时灭活导致快速细胞裂解。基于PBP 1A/PBP 1B组合的必要性，我们推断，可以通过筛选具有PBP 1B损失的合成致死突变体（slb突变体）来鉴定促进PBP 1A活性所需的因子，反之亦然。使用这种方法，我们有牵连的几个已知的分裂蛋白和未知功能的脂蛋白在组装PG的PBP 1A。在本提案的前两个目标中，我们描述了旨在研究PBP 1A与这些因子之间的联系的遗传学、细胞生物学和生物化学实验。这些研究将帮助我们确定Slb因子是否直接与PBP 1A的两种酶活性相互作用和/或影响其中之一。在相关工作中，我们发现EnvC蛋白可能是PG水解酶（酰胺酶）AmiA和AmiB的激活剂，其刺激它们的活性以在胞质分裂期间引起子细胞分离。具体目标3旨在确定EnvC和酰胺酶如何合作以执行这种微妙的操作，而不会导致PG层的致命破坏。我们将开始通过定义EnvC可能激活酰胺酶的机制和识别这种激活活性的调节剂来解决这个问题。我们的工作的长期目标是发展的PG组装的PBPs的分子理解，以及它是如何被改造的PG水解酶在一个受控的方式。通过获得这种理解，我们希望发现新的方法来破坏PG合成和水解之间的细胞平衡，以开发新型的裂解抗生素。 公共卫生相关性：细菌细胞通常由一层称为肽聚糖的坚韧聚合物网络强化。肽聚糖合成途径是一个极好的抗生素靶点，因为这一层是细菌所独有的，对细菌的完整性至关重要。这项研究计划的重点是识别和表征构建肽聚糖层的全套细胞因子，以帮助我们发现其他方法来破坏未来开发新抗生素疗法的途径。

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. PUBLIC HEALTH RELEVANCE: Bacterial cells are typically fortified by a layer of tough polymer meshwork called peptidoglycan. The pathway for peptidoglycan synthesis an excellent antibiotic target because this layer is unique to bacteria and essential for their integrity. The focus of this research proposal is to identify and characterize the full set of cellular factors that build the peptidoglycan layer to help us uncover additional ways to disrupt the pathway for the future development of new antibiotic therapies.