Peptidoglycan Biogenesis in Escherichia Coli

描述（由申请人提供）：大多数细菌周围都有一种叫做肽聚糖（PG）的交联多糖聚合物，这对维持细胞形状和完整性至关重要。由于它的重要性、表面暴露和细菌的独特性，PG合成途径一直是我们许多最重要的抗菌治疗的有效靶点，如青霉素和万古霉素。青霉素靶向PG合成酶，称为青霉素结合蛋白（PBPs）。这些酶有多种类型，但主要的细胞PG合成酶被认为是双功能PBPs，因为它们分别具有合成PG聚糖链和交联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，但我们仍然不了解双功能PBPs如何组装细胞形状的PG网络，或者哪些其他因素可能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，以大肠杆菌作为模型系统来识别体内适当PBP功能所需的因素。大肠杆菌编码三种双功能PBPs: PBP1A、PBP1B和PBP1C。每一个都是单独不可缺少的，但PBP1A和PBP1B同时失活导致细胞快速裂解。基于PBP1A/PBP1B组合的重要性，我们推断促进PBP1A活性所需的因素可以通过筛选随着PBP1B的丧失而合成致死的突变体（slb突变体）来确定，反之亦然。利用这种方法，我们发现PBP1A在PG的组装过程中有几种已知的分裂蛋白和一种功能未知的脂蛋白。在本提案的前两个目的中，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，在细胞分裂过程中导致子细胞分离。特异性目标3旨在确定EnvC和酰胺酶如何合作完成这样一个微妙的操作，而不会导致PG层的致命破坏。我们将通过定义EnvC激活酰胺酶的机制和识别这种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通过PBPs对PG组装进行分子理解，以及如何通过PG水解酶以受控的方式进行重塑。通过获得这一认识，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，以开发新型的溶解抗生素。

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.