Peptidoglycan Biogenesis in Escherichia Coli

描述（由申请人提供）：大多数细菌细胞被由肽聚糖（PG）制成的细胞壁基质包围。这一外骨骼层加强细胞膜抵抗内部渗透压，对细胞完整性至关重要。由于PG是青霉素、万古霉素等许多广泛应用的抗生素的靶点，因此了解PG的生物发生机制仍然具有重要的现实意义。随着美国和国外抗生素耐药感染发生率的上升，现在比以往任何时候都更重要的是发现PG组装等基本途径的新漏洞，以便可以利用它们开发下一代抗菌疗法。在这方面，多年来大多数关于PG生物发生的研究都集中在PG合成酶青霉素结合蛋白（PBPs）的活性上，这是青霉素的靶标。然而，近年来，人们已经清楚地认识到，能够在PG网络中切割键的酶对于PG的正常生物发生同样重要。这些所谓的PG水解酶是潜在的危险酶。如果不加以控制，它们会破坏细胞壁并诱导细胞裂解。因此，控制PG水解酶的调控机制为新型裂解诱导抗生素提供了有吸引力的靶点。为了更好地理解这些控制，我们在之前的资助期集中在鉴定控制PG水解酶的调节因子上，重点是革兰氏阴性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位点的调节结构域自抑制。为了促进细胞分裂，我们发现这些因子需要由具有LytM结构域的分裂蛋白EnvC和NlpD激活。我们进一步发现EnvC对酰胺酶的激活是由abc转运蛋白样复合物FtsEX控制的，这表明FtsEX可能是一种构象调节剂，利用核苷酸结合和水解的调节能力来控制细胞表面PG的裂解。尽管这些发现代表了我们对PG水解酶调控的理解取得了很大的进展，但LytM因子激活酰胺酶的确切机制以及FtsEX对PG水解酶活性的控制仍不清楚。目前还不清楚这些系统催化的PG重塑事件如何与分裂装置的其他主要活动适当协调。因此，揭示这些机制将是下一个筹资期的一个主要目标。我们还将利用我们在酰胺酶调控研究方面的专业知识来研究PG水解酶的功能和调控，这些酶参与PG生物发生的其他重要方面。拟议的实验将建立在初始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型生物学机制。

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.