Peptidoglycan Biogenesis in Escherichia Coli

大肠杆菌中的肽聚糖生物合成

• 批准号: 8815765
• 负责人: Thomas G Bernhardt
• 金额: $ 44.7万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8815765
• 关键词: ATP-Binding Cassette Transporters; Active Sites; Address; Amidohydrolases; Animal Model; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Bacillus subtilis; Bacteria; Binding; Biochemical; Biochemical Genetics; Biogenesis; Biological; Biological Assay; Cell Wall; Cell division; Cell membrane; Cell surface; Cells; Cleaved cell; Complex; Coupling; Cytokinesis; Cytolysis; Development; Elements; Enzymes; Escherichia coli; Event; Foundations; Funding; Future; Genetic; Genetic study; Goals; Gram-Negative Bacteria; Hydrolysis; Incidence; Infection; Left; Link; Lipoproteins; Lytic; Membrane; N-Acetylmuramoyl-L-alanine Amidase; Names; Nucleotides; Osmotic Pressure; Pathway interactions; Penicillin-Binding Proteins; Penicillins; Peptidoglycan; Pharmaceutical Preparations; Physiological; Polysaccharides; Positioning Attribute; Process; Proteins; Regulation; Research Proposals; Role; Site; Streptococcus pneumoniae; Structure; System; Tertiary Protein Structure; United States; Vancomycin; Work; amidase; enzyme activity; exoskeleton; genetic analysis; in vivo; innovation; insight; interest; next generation; novel; public health relevance; research study; structural biology; synthetic enzyme

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.

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。该外骨骼层增强细胞膜抵抗内部渗透压的能力，对于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实际意义。随着美国和国外抗生素耐药性感染发生率的上升，现在比以往任何时候都更重要的是发现 PG 组装等基本途径中的新漏洞，以便将它们用于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称为青霉素结合蛋白 (PBP) 的 PG 合成酶的活性上，PBP 是青霉素的靶标。然而，近年来，人们已经清楚，能够裂解 PG 网络中的键的酶对于适当的 PG 生物发生同样重要。这些所谓的 PG 水解酶是具有潜在危险的酶。如果不加以控制，它们会损坏细胞壁并诱导细胞裂解。因此，控制 PG 水解酶的调节机制代表了新型裂解诱导抗生素的有吸引力的目标。为了更好地理解这些控制，我们在之前的资助期间将大部分精力集中在确定控制 PG 水解酶的调节因子上，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型生物，我们发现酰胺酶都受到封闭其活性位点的调节域的自抑制。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（称为 EnvC 和 NlpD）的分裂蛋白激活。我们进一步表明，EnvC 的酰胺酶激活是由 ABC 转运蛋白（如复合体 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的 PG 裂解。尽管这些发现代表了我们对 PG 水解酶调节的理解取得了巨大进展，但 LytM 因子激活酰胺酶和 FtsEX 控制 PG 水解酶活性的精确机制仍不清楚。我们也不知道这些系统催化​​的 PG 重塑事件如何与该部门的其他主要活动适当协调。因此，揭示这些机制将是下一个资助期的主要目标。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参与 PG 生物合成其他重要方面的 PG 水解酶的功能和调节。拟议的实验将建立在初始资助期间奠定的坚实基础上，使我们能够继续揭示与未来抗生素开发相关的新生物机制。