Envelope Biogenesis in Gram-negative Bacteria

革兰氏阴性细菌的包膜生物发生

• 批准号: 9513001
• 负责人: Natividad Ruiz
• 金额: $ 32.26万
• 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/9513001
• 关键词: ATP-Binding Cassette Transporters; 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; 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（由申请方提供）：革兰氏阴性菌的包膜由两个脂质双层（内膜和外膜）界定（分别为IM和OM）。OM的外部小叶含有密集包装的脂多糖（LPS），其对小疏水分子具有异常高的不渗透性。因此，革兰氏阴性菌对许多抗生素具有天然耐药性。IM和OM被称为周质的水隔室分开，周质中存在由肽聚糖组成的细胞壁。肽聚糖细胞壁是保护细胞免受渗透裂解的基本聚合刚性结构。鉴于细胞被膜的结构和保护功能，适当的被膜生物发生对于细菌在许多环境中的生存至关重要。强调这一点的事实是，许多抗生素靶向包膜生物合成途径。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构建其细胞包膜。在这里，我们建议主要使用遗传学和生物化学方法的组合来研究两个高度保守的系统，该系统将糖脂从其合成位点跨细胞包膜转运到它们发挥功能的细胞区室：1）MurJ，一种多位IM蛋白，其促进肽聚糖生物合成中最不为人知的步骤，脂质连接的肽聚糖前体脂质II跨IM的易位;和2）Lpt（LPS转运），一种跨越包膜的多蛋白质桥，其功能是将LPS从IM转运到细胞表面。这两种系统对于许多细菌的生存能力都是必不可少的，包括我们的模式生物大肠杆菌。在目标1中，我们提出研究以理解MurJ用于翻转脂质II的机制，通过：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在运输循环期间经历的构象变化;以及，d）研究MurJ如何被供电。在目标2中，我们将通过将我们的研究集中在LptFGB 2C亚复合物上来研究LPS转运中最不了解的步骤，LptFGB 2C亚复合物是一种独特的ATP结合盒转运蛋白，其为从IM提取LPS及其沿Lpt桥沿着转运至细胞表面提供动力。具体地，在目标2中，我们将：a）确定膜组分LptF和LptG相对于IM的拓扑结构; B）定义LptFGB 2 C亚复合物中的蛋白质-蛋白质相互作用;和c）阐明LptFGB 2 C如何将细胞质中的ATP结合和水解与从IM的外小叶提取LPS偶联。由于MurJ功能的抑制导致细胞溶解，并且Lpt系统中的缺陷可以增加OM对许多抗生素的渗透性，甚至导致死亡，因此从所提出的工作中获得的知识将有助于开发新的抗微生物疗法。特别需要对Lpt进行研究，以了解我们如何克服革兰氏阴性菌对抗生素的先天抗性，因为细胞表面存在LPS所施加的屏障。