Molecular mechanisms required for the maintenance of the gram-negative outer membrane

维持革兰氏阴性外膜所需的分子机制

• 批准号: 10159193
• 负责人: Michael Stephen Trent
• 金额: $ 40.96万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10159193
• 关键词: ATP-binding cassette transport; Antibiotics; Architecture; Bacteria; Binding; Binding Proteins; Biochemical; Biological Assay; Cell Death; Cells; Communicable Diseases; Complex; Data; Detergents; Encapsulated; Environment; Environmental Protection; Environmental Risk Factor; Enzymes; Escherichia coli; Generations; Genetic; Glycerophospholipids; Gram-Negative Bacteria; Growth; Hand; Human; In Vitro; Infection; Innate Immune System; Investigation; Knowledge; Lipid A; Lipid Binding; Lipids; Lipopolysaccharides; Lipoproteins; Maintenance; Membrane; Membrane Lipids; Modification; Molecular; Multi-Drug Resistance; Mutation; Organism; Pathway interactions; Peptidoglycan; Phospholipases A; Phospholipids; Proteins; Reporting; Research; Role; Salmonella; Site; Stress; Structure; Substrate Specificity; Surface; System; Virulence; anterograde transport; antimicrobial; antimicrobial peptide; base; cell envelope; crosslink; environmental stressor; fighting; in vivo; lipophilicity; membrane assembly; migration; monolayer; novel; novel therapeutics; pathogen; pathogenic bacteria; periplasm; prevent; protein protein interaction; retrograde transport

Abstract The bacterial cell envelope is a remarkable and complex structure that guards bacteria from their surrounding environment. A defining feature of gram-negative bacteria is the presence of an outer membrane (OM) that encapsulates the peptidoglycan layer of these organisms. While the inner membrane (IM) is composed of glycerophospholipids (GPLs), the OM is a bilayer with extreme lipid asymmetry with GPL confined to the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane organization affords gram-negative bacteria protection from large polar molecules, as well as lipophilic compounds, serving as an essential innate barrier to a variety of antibiotics. However, once assembled, environmental factors can disrupt the LPS monolayer resulting in shedding of LPS, and as a consequence, migration of GPLs from the inner leaflet to the outer leaflet of the OM. Extensive formation of GPL rafts at the bacterial surface results in the loss of barrier function which leads to cell death. To prevent this, the cell must maintain the OM asymmetry even under extreme environmental stress. The overall objective of this application is to investigate the molecular mechanisms required for maintenance of OM asymmetry, including the role of the recently identified Mla retrograde GPL transport system. We will also investigate two additional systems, Pqi and Yeb systems, that may also serve in GPL transport. All three systems (Mla, Pqi, and Yeb) are highly conserved across Gram-negative bacteria and disruption of OM maintenance machinery has been shown to result in decreased virulence for many pathogens. The Specific Aims of the current proposal are: (1) structure and functional analysis of Mla lipid binding proteins; (2) overall architecture and protein-protein interactions of Mla components; and (3) investigation of lipid binding by GPL transport systems in whole bacteria. Completion of the Aims will fill major gaps towards understanding maintenance of OM asymmetry and provide new avenues for the generation of novel antimicrobials.

摘要 细菌的细胞被膜是一种非凡而复杂的结构，它保护细菌免受它们的 周边环境革兰氏阴性菌的一个明显特征是存在外膜 (OM)包裹着这些生物体的肽聚糖层。内膜（IM） OM由甘油磷脂（GPL）组成，是一种与GPL极不对称的双层脂质 局限于内小叶，脂多糖（LPS）局限于外小叶。这种独特的膜 组织提供革兰氏阴性菌保护免受大极性分子，以及亲脂性 化合物，作为一个基本的先天屏障，以各种抗生素。 然而，一旦组装，环境因素可以破坏LPS单层，导致LPS脱落。 LPS，并因此导致GPL从OM的内瓣叶迁移至外瓣叶。广泛 GPL筏在细菌表面的形成导致屏障功能的丧失，从而导致细胞死亡。 为了防止这种情况，即使在极端的环境压力下，细胞也必须保持OM不对称性。的 本申请的总体目标是研究维持 OM不对称性，包括最近发现的Mla逆行GPL运输系统的作用。我们还将 研究另外两个系统，Pqi和Yeb系统，它们也可能用于GPL传输。所有三 系统（Mla，Pqi和Yeb）在革兰氏阴性菌中高度保守，OM破坏 已显示维持机制导致许多病原体的毒力降低。具体 本研究的主要目的是：（1）Mla脂质结合蛋白的结构和功能分析;（2）Mla脂质结合蛋白的整体结构和功能分析;（3）Mla脂质结合蛋白的结构和功能分析;（4）Mla脂质结合蛋白的结构和功能分析。 Mla组分的结构和蛋白质-蛋白质相互作用;（3）通过GPL研究脂质结合 整个细菌的运输系统。目标的完成将填补理解方面的主要空白 维持OM不对称性，并为产生新型抗菌剂提供新的途径。