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

摘要 细菌的细胞膜是一种非凡而复杂的结构，它保护细菌免受 周围的环境。革兰氏阴性细菌的一个显著特征是外膜的存在。 (OM)包裹这些生物体的肽聚糖层。而内膜(IM)是 OM是由甘油磷脂(GPL)组成的双层，与GPL具有极大的脂质不对称性 局限于内叶，而脂多糖(LPS)定位于外叶。这种独特的膜 该组织为革兰氏阴性细菌提供保护，使其免受大极性分子的伤害，并具有亲脂性 化合物，作为一种基本的先天屏障，对各种抗生素。 然而，一旦组装，环境因素可以破坏内毒素单层，导致脱落 因此，GPL从OM的内叶迁移到外叶。广泛性 在细菌表面形成GPL筏会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的不对称性。这个 这项应用的总体目标是研究维持心脏功能所需的分子机制。 OM的不对称性，包括最近发现的MLA逆行GPL运输系统的作用。我们还将 调查另外两个系统，PQI和YEB系统，这两个系统也可能在GPL传输中服务。三个都是 系统(MLA、PQI和YEB)在革兰氏阴性菌和OM的破坏中高度保守 维修机械已被证明能降低许多病原体的致病力。具体的 本提案的目的是：(1)MLA脂结合蛋白的结构和功能分析；(2)总体 MLA组分的结构和蛋白质-蛋白质相互作用；(3)GPL对脂类结合的研究 整个细菌中的运输系统。这些目标的完成将填补理解方面的主要空白 维持OM的不对称性，并为产生新的抗菌剂提供新的途径。

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.