Biogenesis and maintenance of the outer membrane of Gram-negative bacteria

革兰氏阴性菌外膜的生物发生和维持

• 批准号: 10477940
• 负责人: Thomas J. Silhavy
• 金额: $ 80.33万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10477940
• 关键词: Antibiotics; Architecture; Biogenesis; Biological Models; Cell surface; Cells; Collection; Combined Modality Therapy; Cytoplasm; Defect; DegP protease; Diffuse; Environment; Enzymes; Escherichia coli; Face; Funding; Genes; Goals; Gram-Negative Bacteria; Island; Knowledge; Ligase; Lipid Bilayers; Lipids; Lipopolysaccharides; Lipoproteins; Maintenance; Membrane; Modeling; Molecular; Molecular Chaperones; Mutation; Nature; O Antigens; Pathway interactions; Peptides; Periodicity; Phenotype; Phospholipids; Physiology; Process; Proteins; Role; Surface; Testing; Work; aqueous; biological adaptation to stress; cell envelope; cell killing; enterobacterial common antigen; insight; membrane biogenesis; mutant; new therapeutic target; novel; novel antibiotic class; periplasm; protein protein interaction; response; sortase; tool; trafficking; translational study

PROJECT SUMMARY The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and an aqueous compartment termed the periplasm that is located between them. A long-term goal of my lab has always been to understand the mechanisms of envelope biogenesis using Escherichia coli as a model system. This proposal concerns OM biogenesis and the stress responses that maintain cell envelope physiology. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS), lipoproteins, and β-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified the essential proteins required to assemble LPS (LptABCDEFG) and OMPs (BamABCDE) in the OM and we have provided evidence of a diffusive mechanism of phospholipid transport between the IM and the OM. In the current funding period, we have shown that the conditional lethal phenotype of bamB bamE double mutants can be suppressed simply by deleting a surface-exposed lipoprotein, we revealed the existence of an alternate lipoprotein trafficking pathway, we uncovered a role for the cyclic form of Enterobacterial Common Antigen in maintaining the OM barrier, and we identified mutations that activate or prime the σE stress response that suppress a variety of OMP and Bam defects. In translational studies, we used our knowledge of OM biogenesis to discover a new class of antibiotics that work to inhibit BamA at the cell surface. We propose to use our large collection of mutations that alter the Bam components or various OMP substrates together with our collection of suppressors as tools to probe the OMP assembly process. In particular, we will probe the function of the non-essential BamBCE lipoproteins and test our hypothesis that BamD does not perform a truly essential mechanistic role, but rather functions as a regulator to control the activity of BamA. We will test the role of the chaperone Skp as a specific adaptor for the periplasmic protease DegP. We also posit that the trimeric nature of the major OMPs functions as a global organizer of OM architecture by providing multiple interacting faces to allow the protein-protein interactions necessary for the formation of protein islands. Our studies on LPS assembly will utilize a mutant O-antigen ligase and the enzyme sortase to attach peptides or proteins to LPS to challenge the capabilities of the LptDE translocon. We will also test our model that three essential IM proteins, YejM, YciM, and FtsH comprise a novel pathway that regulates LPS synthesis in response to the lipid status of the OM. The mlaA* mutation destabilizes the OM by increasing LPS levels. This causes membrane loss by OM vesiculation and IM PLs flow into the OM to replace the loss. We have identified a mutation that slows this lipid flow and we believe that continued study of this gene may provide insights into the poorly understood process of anterograde PL transport.

项目概要 革兰氏阴性菌的细胞膜含有两层膜，内膜（IM）和外膜（OM），以及一层膜。 位于它们之间的水室称为周质，这是我实验室的长期目标。 使用大肠杆菌作为模型系统来了解包膜生物发生的机制。 该提案涉及 OM 生物发生和维持细胞包膜生理学的应激反应。 OM、磷脂 (PL)、脂多糖 (LPS)、脂蛋白和 β-桶蛋白的成分 (OMP)，在细胞质或 IM 的内部小叶中合成，我们已经鉴定了必需的蛋白质。 需要在 OM 中组装 LPS (LptABCDEFG) 和 OMP (BamABCDE)，并且我们已提供证据 IM 和 OM 之间磷脂运输的扩散机制。 我们已经证明bamB bamE双突变体的条件致死表型可以简单地被抑制 通过删除表面暴露的脂蛋白，我们揭示了替代脂蛋白运输的存在 途径，我们发现了肠杆菌共同抗原的环状形式在维持 OM 中的作用 屏障，我们发现了激活或引发 σE 应激反应的突变，从而抑制各种 OMP 在转化研究中，我们利用 OM 生物发生的知识发现了一类新的缺陷。 抑制细胞表面 BamA 的抗生素。 我们建议使用我们大量的突变来改变 Bam 成分或各种 OMP 底物 特别是，我们将与我们收集的抑制器一起作为探测 OMP 组装过程的工具。 探究非必需 BamBCE 脂蛋白的功能并检验我们的假设，即 BamD 不 发挥真正重要的机械作用，而是作为调节器来控制 BamA We 的活动。 我们还假设伴侣蛋白 Skp 作为周质蛋白酶 DegP 的特异性接头的作用。 主要 OMP 的三聚性质通过提供 OM 架构的全球组织者功能 多个相互作用的面，以允许形成蛋白质岛所需的蛋白质-蛋白质相互作用。 我们对 LPS 组装的研究将利用突变型 O 抗原连接酶和酶分选酶来连接肽 或 LPS 蛋白来挑战 LptDE 易位子的能力 我们还将测试这三个模型。 必需的 IM 蛋白 YejM、YciM 和 FtsH 构成了一条调节 LPS 合成的新途径 OM 的脂质状态。 mlaA* 突变通过增加 LPS 水平来破坏 OM 的稳定性，这会导致 OM 膜损失。 囊泡形成和 IM PL 流入 OM 以替代损失。我们已经发现了一种可以减缓这种脂质损失的突变。 我们相信，对该基因的持续研究可能会为人们了解甚少的过程提供见解 顺行 PL 运输。