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(申请人提供)：革兰氏阴性菌的包膜由两层脂类双层分隔，内膜和外膜(分别为IM和OM)。OM的外部小叶含有紧密堆积的脂多糖(LPS)，使其对疏水小分子具有异常高的不渗透性。因此，革兰氏阴性细菌对许多抗生素都有天然的抗药性。IM和OM被称为周质的水室隔开，胞壁由肽聚糖组成。肽聚糖细胞壁是一种基本的聚合物刚性结构，保护细胞免受渗透溶解。鉴于细胞被膜的结构和保护功能，适当的被膜生物发生对细菌在许多环境中的生存至关重要。突显这一点的是，许多抗生素针对的是包膜生物发生途径。我们的长期目标是在分子水平上了解革兰氏阴性细菌是如何形成细胞膜的。在这里，我们建议主要使用遗传和生化方法相结合的方法来研究两个高度保守的系统，它们将糖脂从其合成部位运输到细胞隔室，在那里它们发挥作用：1)Murj，一种多位IM蛋白，它促进了肽聚糖生物合成中最鲜为人知的步骤，即脂联肽多糖前体脂质II的跨IM转运；以及2)LPT(脂多糖转运)，一种跨越被膜的多蛋白质桥，其功能是将LPS从IM运输到细胞表面。这两个系统对许多细菌的生存都是必不可少的，包括我们的模式生物大肠杆菌。在目标1中，我们建议通过以下几个方面来研究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在运输周期中经历的构象变化；以及d)研究Murj是如何被驱动的。在目标2中，我们将通过重点研究LptFGB2C亚复合体来研究内毒素转运中最鲜为人知的步骤，LptFGB2C亚复合体是一种独特的ATP结合盒转运体，它为从IM中提取内毒素并将其沿着LPT桥运输到细胞表面提供动力。具体地说，在目标2中，我们将：a)确定膜成分LptF和LptG相对于IM的拓扑结构；b)定义LptFGB2C亚复合体中的蛋白质-蛋白质相互作用；以及c)阐明LptFGB2C如何将细胞质中的ATP结合和水解与IM外层小叶中的脂多糖的提取结合起来。由于MURJ功能的抑制会导致细胞溶解，而LPT系统中的缺陷可以增加OM对许多抗生素的通透性，甚至导致死亡，因此从拟议的工作中获得的知识将有助于开发新的抗菌疗法。尤其需要对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耐药性，因为细胞表面存在内毒素造成的屏障。