Synthesis and transport of outer membrane components across the Gram-negative cell envelope

摘要 抗生素耐药性的增加和新抗菌素发现的减少威胁着全球 医疗保健。特别令人担忧的是革兰氏阴性病原体，即具有额外外膜的生物体 (OM)对多种抗生素具有内在耐药性。与内膜(IM)不同的是 仅由甘油磷脂(GPLs)组成的OM与内叶中的GPLs不对称 脂多糖(LPS)定位于外叶。这种独特的膜不对称性提供了 保护不受大极性分子和亲脂化合物的伤害，形成不透水屏障。自.以来 OM是必不可少的，其组装所需的途径是抗菌设计的关键靶点。目前， 临床上没有直接针对OM生物发生的抗生素。因此，调查仍然至关重要。 用于未来和当前抗菌设计的细胞包膜生物发生。 在过去的几十年里，我们扩大了对OM组装的理解，揭示了新的目标 抗菌设计。然而，仍然存在一个重大差距。如何将GPL从IM传输到OM 穿过房水周质？最近，我们发现Asma样家族(YhdP， TAMB和YDBH)对OM完整性至关重要，并参与GPL运输。我们发现YhdP、TAMB和 YDBH在OM脂稳态中的作用是多余的；然而，它们并不等同。值得注意的是，这三个人 蛋白质与真核细胞的GPL转运蛋白具有相同的同源性和结构特征，并且能够跨越 周质。这项应用的总体目标是研究所需的分子机制 用于革兰氏阴性OM的组装和维护。更具体地说，我们将描述路径 以大肠杆菌为模型系统，通过细胞膜运输GPL所需的。在当前 应用我们将(I)描述主要的GPL转运体(YhdP、TAMB和YDBH)，(Ii)识别附件 GPL跨双层运动所需的蛋白质，以及(Iii)确定这些系统的损失如何影响OM 脂平衡与抗生素耐药性。

Abstract The increasing rise in antibiotic resistance and the diminished discovery of new antimicrobials threatens global healthcare. Of particular concern are Gram-negative pathogens, organisms with an additional outer membrane (OM) that provides intrinsic resistance to multiple classes of antibiotics. Unlike the inner membrane (IM) that is composed solely of glycerophospholipids (GPLs), the OM is asymmetrical with GPLs found in the inner leaflet and lipopolysaccharide (LPS) localized to the outer leaflet. This unique membrane asymmetry affords protection from large polar molecules, as well as lipophilic compounds, creating an impervious barrier. Since the OM is essential, pathways required for its assembly are key targets for antimicrobial design. Currently, there are no antibiotics that directly target OM biogenesis in clinical use. Thus, it remains critical to investigate cell envelope biogenesis for future and current antimicrobial design. Over the last few decades, we have expanded our understanding of OM assembly revealing new targets for antimicrobial design. However, one major gap remained. How are GPLs transported from the IM to the OM across the aqueous periplasm? Recently, we discovered that key members of the AsmA-like family (YhdP, TamB, and YdbH) are critical for OM integrity and involved in GPL transport. We found that YhdP, TamB, and YdbH are redundant in their role in OM lipid homeostasis; however, they are not equivalent. Notably, all three proteins share homology and structural features with eukaryotic GPL transporters and are capable of spanning the periplasm. The overall objective of this application is to investigate the molecular mechanisms required for the assembly and maintenance of the Gram-negative OM. More specifically, we will characterize pathways required to transport GPLs across the cell envelope using E. coli as the model system. In the current application we will (i) characterize the major GPL transporters (YhdP, TamB, and YdbH), (ii) identify accessory proteins required for GPL transbilayer movement, and (iii) determine how loss of these systems impact OM lipid homeostasis and antibiotic resistance.