Peptidoglycan Biogenesis in Escherichia Coli

描述(申请人提供)：大多数细菌细胞被由肽聚糖(PG)组成的细胞壁基质包围。这一外骨骼层加强了细胞膜抵抗内渗透压力的能力，对细胞的完整性至关重要。由于它是许多广泛使用的抗生素如青霉素和万古霉素的靶标，因此了解PG的生物发生仍然具有重要的现实意义。随着抗生素耐药感染在美国和国外的发病率不断上升，现在比以往任何时候都更加重要的是，在PG组装等关键途径中发现新的漏洞，以便利用这些漏洞开发下一代抗菌疗法。在这方面，多年来对PG生物发生的研究大多集中在被称为青霉素结合蛋白(PBPS)的PG合成酶的活性上，而PBPS是青霉素的靶标。然而，近年来，很明显，能够裂解PG网络中的键的酶对于正确的PG生物发生同样重要。这些所谓的PG水解酶是潜在的危险酶。如果不加以控制，它们可能会破坏细胞壁并诱导细胞溶解。因此，控制PG水解酶的调控机制是新型裂解诱导抗生素的诱人靶点。为了更好地了解这些控制，我们将前一个资金阶段的大部分时间集中在确定PG水解酶的调控因子上，重点是革兰氏阴性细菌细胞分裂所需的细胞壁酰胺酶。使用大肠杆菌作为我们的模式生物，我们发现酰胺酶都被一个封闭其活性部位的调节结构域所抑制。为了促进细胞分裂，我们发现这些因子需要通过带有LytM结构域的分裂蛋白EnvC和NLPD来激活。我们进一步证明了EnvC对酰胺酶的激活是由类似ABC转运蛋白的复合体FtsEX控制的，这表明FtsEX可能是一个令人兴奋的构象调节因子，它利用核苷酸结合和水解的调节力来控制细胞表面的PG切割。尽管这些发现标志着我们对PG水解酶调控的理解有了很大的进步，但LytM因子激活酰胺酶和FtsEX控制PG水解酶活性的确切机制仍不清楚。由这些系统催化的PG重塑事件如何与师器官的其他主要活动适当地协调也是未知的。因此，发现这些机制将是下一个供资阶段的主要目标。我们还将利用我们在酰胺酶调节研究方面的专业知识来研究PG水解酶在PG生物发生的其他重要方面所涉及的功能和调节。拟议的实验将建立在最初的资助期奠定的坚实基础上，并使我们能够继续揭示与未来抗生素开发相关的新的生物学机制。

DESCRIPTION (provided by applicant): Most bacterial cells are surrounded with a cell wall matrix made of peptidoglycan (PG). This exoskeletal layer fortifies the cell membrane against internal osmotic pressure and is essential for cell integrity. Because it is the target for many widely used antibiotics like penicillin and vancomycin, understanding the biogenesis of PG continues to be of great practical significance. With the rising incidence of antibiotic resistant infections in the United States and abroad, it is now more important than ever to discover new vulnerabilities in essential pathways like PG assembly so that they can be exploited for the development of next generation antibacterial therapies. In this regard, most studies of PG biogenesis over the years have focused on the activities of PG synthase enzymes called penicillin-binding proteins (PBPs), the targets of penicillin. In recent years, however, it has become clear that enzymes capable of cleaving bonds in the PG network are equally important for proper PG biogenesis. These so-called PG hydrolases are potentially dangerous enzymes. If left unchecked, they can damage the cell wall and induce cell lysis. Thus, the regulatory mechanisms controlling PG hydrolases represent attractive targets for new classes of lysis-inducing antibiotics. To better understand these controls, we focused much of the previous funding period on identifying regulators governing PG hydrolases with an emphasis on cell wall amidases required for cell division in gram-negative bacteria. Using Escherichia coli as our model organism, we discovered that the amidase enzymes are all autoinhibited by a regulatory domain that occludes their active site. To promote cell division, we found that these factors require activation by division proteins with LytM domains called EnvC and NlpD. We further showed that amidase activation by EnvC is controlled by the ABC-transporter like complex FtsEX, suggesting the exciting possibility that FtsEX may be a conformational regulator that uses the regulatory power of nucleotide binding and hydrolysis to control PG cleavage at the cell surface. Although these discoveries represent great progress in our understanding of PG hydrolase regulation, the precise mechanisms of amidase activation by the LytM factors and the control of PG hydrolase activity by FtsEX remain unclear. It is also not known how the PG remodeling events catalyzed by these systems are properly coordinated with other major activities of the division apparatus. Uncovering these mechanisms will therefore be a major goal of the next funding period. We will also leverage our expertise in studies of amidase regulation to investigate the function and regulation of PG hydrolases involved in other important aspects of PG biogenesis. The proposed experiments will build on the strong foundation laid in the initial funding period and allow us to continue revealing novel biological mechanisms relevant to future antibiotic development.