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.

抽象的 细菌细胞膜是一种非凡而复杂的结构，可以保护细菌免受细菌侵害。 周边环境。革兰氏阴性细菌的一个决定性特征是存在外膜 （OM）封装这些生物体的肽聚糖层。虽然内膜 (IM) 是 OM 由甘油磷脂 (GPL) 组成，是与 GPL 具有极端脂质不对称性的双层 限制于内叶，脂多糖（LPS）定位于外叶。这种独特的膜 组织为革兰氏阴性菌提供保护，使其免受大极性分子以及亲脂性分子的侵害 化合物，作为多种抗生素的重要先天屏障。 然而，一旦组装，环境因素可能会破坏 LPS 单层，导致 LPS 脱落 LPS，以及因此导致的 GPL 从 OM 的内部小叶迁移到外部小叶。广泛的 在细菌表面形成 GPL 筏会导致屏障功能丧失，从而导致细胞死亡。 为了防止这种情况发生，即使在极端的环境压力下，细胞也必须保持 OM 不对称性。这 该应用程序的总体目标是研究维持 OM 不对称，包括最近确定的 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。全部三个 系统（Mla、Pqi 和 Yeb）在革兰氏阴性菌和 OM 破坏中高度保守 维护机制已被证明可以降低许多病原体的毒力。具体 当前提案的目的是：（1）Mla脂质结合蛋白的结构和功能分析； (2)总体 Mla 成分的结构和蛋白质-蛋白质相互作用； (3) GPL 脂质结合研究 整个细菌中的运输系统。目标的完成将填补理解方面的重大差距 维持 OM 不对称性并为新型抗菌药物的产生提供新途径。