Envelope Biogenesis in Gram-negative Bacteria

革兰氏阴性细菌的包膜生物发生

• 批准号: 9302818
• 负责人: Natividad Ruiz
• 金额: $ 32.29万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-05 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9302818
• 关键词: ATP-Binding Cassette Transporters; Alpha Cell; Anabolism; Animal Model; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biochemical; Biogenesis; Cations; Cause of Death; Cell Membrane Permeability; Cell Shape; Cell Survival; Cell Wall; Cell physiology; Cell surface; Cells; Complex; Couples; Crude Extracts; Cytolysis; Cytoplasm; Defect; Development; Disaccharides; Environment; Escherichia coli; Funding; Genetic; Glycolipids; Goals; Gram-Negative Bacteria; Growth; Hydrolysis; Hydrophobicity; Immune system; Knowledge; Link; Lipid Bilayers; Lipids; Lipopolysaccharides; Mediating; Membrane; Membrane Proteins; Molecular; Molecular Conformation; Organism; Pathogenesis; Pathway interactions; Peptidoglycan; Permeability; Polymers; Proteins; Resistance; Side; Site; Structure; System; Testing; Transmembrane Transport; Travel; Work; antimicrobial; aqueous; cell envelope; design; environmental change; extracellular; global health; in vivo; killings; microorganism; muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; novel; pathogen; periplasm; protein protein interaction; public health relevance; scaffold; small molecule

 DESCRIPTION (provided by applicant): The envelope of Gram-negative bacteria is delimited by two lipid bilayers, the inner and outer membranes (IM and OM, respectively). The external leaflet of the OM contains densely packed lipopolysaccharides (LPS) that confer unusually high impermeability towards small hydrophobic molecules. As a result, Gram-negative bacteria are naturally resistant to many antibiotics. The IM and OM are separated by the aqueous compartment known as the periplasm where a cell wall composed of peptidoglycan resides. The peptidoglycan cell wall is an essential polymeric rigid structure that protects cells from osmotic lysis. Given the structural and protective functions of the cell envelope, proper envelope biogenesis is crucial for the survival of bacteria in many environments. Underscoring this is the fact that many antibiotics target envelope biogenesis pathways. Our long-term goal is to understand at the molecular level how Gram-negative bacteria build their cell envelope. Here, we propose to primarily use a combination of genetic and biochemical approaches to investigate two highly conserved systems that transport glycolipids across the cell envelope from their site of synthesis to the cellular compartment where they function: 1) MurJ, a polytopic IM protein that facilitates the most poorly understood step in peptidoglycan biosynthesis, the translocation of the lipid-linked peptidoglycan precursor lipid II across the IM; and 2) Lpt (LPS transport), a mult-protein bridge that spans the envelope and that functions to transport LPS from the IM to the cell surface. Both of these systems are essential for the viability of many bacteria including our model organism Escherichia coli. In aim 1, we propose studies to understand the mechanism that MurJ uses to flip lipid II by: a) conducting structure-function studies on MurJ; b) determinin how MurJ interacts with lipid II; c) probing conformational changes that MurJ undergoes during the transport cycle; and, d) studying how MurJ is powered. In aim 2, we will investigate the most poorly understood step in LPS transport by focusing our studies on the LptFGB2C sub-complex, a unique ATP- binding cassette transporter that powers the extraction of LPS from the IM and its transport along the Lpt bridge to the cell surface. Specifically, in aim 2, we will: a) determie the topology of the membrane components LptF and LptG with respect to the IM; b) define protein-protein interactions in the LptFGB2C sub- complex; and c) elucidate how LptFGB2C couples ATP binding and hydrolysis in the cytoplasm to the extraction of LPS from the outer leaflet of the IM. Because inhibition of MurJ function leads to cell lysis and defects in the Lpt system can either increase OM permeability to many antibiotics or even cause death, knowledge gained from the proposed work will help in developing novel antimicrobial therapies. Studies on Lpt are especially needed to understand how we can overcome the innate resistance to antibiotics that Gram- negative have because of the barrier imposed by the presence of LPS at the cell surface.

 描述（由适用提供）：革兰氏阴性细菌的包膜由两个脂质双层（分别为IM和OM）界定。 OM的外部小叶含有纯净的脂多糖（LPS），这些含糖（LPS）很少向小疏水分子赋予高度不渗透性。结果，革兰氏阴性细菌自然对许多抗生素具有抗性。 IM和OM被称为外围室的水室分开，其中由肽聚糖住宅组成的细胞壁。肽聚糖细胞壁是一种必不可少的聚合物刚性结构，可保护细胞免受渗透裂解。鉴于细胞包膜的结构和受保护功能，适当的包膜生物发生对于在许多环境中细菌的存活至关重要。强调这是许多抗生素靶向包膜生物发生途径的事实。我们的长期目标是在分子水平上了解革兰氏阴性细菌如何建立其细胞包膜。 Here, we propose to primary use a combination of genetic and biochemical approaches to investigate two highly constituted systems that transport glycolipids across the cell envelope from their site of synthesis to the cellular compartment where they function: 1) MurJ, a polytopic IM protein that facilitates the most poorly understand step in pepperydoglycan biosynthesis, the translocation of the lipid-linked跨IM的肽聚糖前体脂质II； 2）LPT（LPS Transport），这是一种跨膜的多蛋白桥，并且该功能可将LP从IM传输到细胞表面。这两个系统对于包括我们的模型生物大肠杆菌在内的许多细菌的生存能力都是必不可少的。在AIM 1中，我们提出研究，以了解MURJ用来翻转脂质II的机制：a）对MURJ进行结构 - 功能研究； b）确定MURJ如何与脂质II相互作用； c）探测MURJ在运输周期中发生的构象变化；并且，d）研究MURJ是如何动力的。在AIM 2中，我们将通过将我们的研究集中在LPTFGB2C亚复合物上来研究LPS运输中最鲜为人知的步骤，LPTFGB2C子复合物是一种独特的ATP结合盒式转运蛋白，可以为LPS从IM及其运输沿LPT桥从LPT桥上提取到细胞表面提供动力。具体而言，在AIM 2中，我们将：a）确定相对于IM的膜成分LPTF和LPTG的拓扑； b）在LPTFGB2C亚复合物中定义蛋白质 - 蛋白质相互作用； c）阐明了LPTFGB2C夫妻如何在细胞质中结合和水解与从IM外叶提取LPS的水解。由于对MURJ功能的抑制会导致细胞裂解和LPT系统中的缺陷可以增加对许多抗生素的渗透性，甚至可以导致死亡，因此从提出的工作中获得的知识将有助于开发新的抗菌治疗。特别需要对LPT进行研究，以了解我们如何克服对抗生素的先天抗性，因为LPS在细胞表面存在LPS所施加的障碍。