Peptidoglycan Biogenesis in Escherichia Coli

描述(申请人提供)：大多数细菌被一种称为多肽(PG)的交联型多糖聚合物包围，这种聚合物对维持细胞形状和完整性至关重要。由于其重要性、表面暴露和对细菌的独特性，PG合成途径历来是许多最重要的抗菌治疗的有效靶点，如青霉素和万古霉素。青霉素针对的是被称为青霉素结合蛋白(PBPS)的PG合成酶。这些酶有几个种类，但主要的细胞PG合成酶被认为是双功能的PBPs，因为它们分别具有合成PG的糖链和交联剂所需的转糖基酶和转肽酶活性。尽管它们是重要的抗生素靶点，但我们仍然不知道双功能PBP是如何组装细胞形状的PG网络的，也不知道还有什么额外的因素可以帮助它们完成这一任务。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学方法，以大肠杆菌作为模型系统来确定体内正确的PBP功能所需的因素。大肠杆菌编码三种双功能PbP：PbP1a、PbP1B和PbP1C。每一个都是单独的可有可无的，但同时灭活PBP1A和PBP1B会导致快速的细胞裂解。基于PBP1A/PBP1B结合的重要性，我们推测，通过筛选具有PBP1B缺失的综合致死突变体(SLB突变体)，可以确定促进PBP1A活性所需的因子，反之亦然。利用这种方法，我们已经将几个已知的分裂蛋白和一个未知功能的脂蛋白牵连到PBP1A组装PG的过程中。在这项建议的前两个目标中，我们描述了旨在调查PBP1A和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的两种酶活性中的任何一种相互作用和/或影响。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，在胞质分裂过程中刺激它们的活性，导致子细胞分离。具体目标3试图确定EnvC和amidase如何合作执行如此精细的操作，而不会导致PG层的致命破坏。我们将通过定义EnvC激活酰胺酶的机制并确定这种激活活动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PG组装的分子理解，以及PG水解酶如何以受控的方式重塑PG组装。通过获得这一理解，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，以开发新的裂解类抗生素。

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.