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.

项目摘要 革兰氏阴性细菌的细胞包膜包含两种机制，即内部（IM）和外部（OM），一个机制 水舱称为它们之间的周期。我实验室的长期目标一直 我们将使用大肠杆菌作为模型系统了解包膜生物发生的机制。这 建议涉及OM生物发生和维持细胞包膜生理的压力反应。全部 OM，磷脂（PL），脂多糖（LPS），脂蛋白和β-桶蛋白的成分 （OMP）在IM的细胞质或内部小叶中合成。我们已经确定了必需蛋白 在OM中组装LPS（LPTABCDEFG）和OMP（BAMABCDE）所需 IM和OM之间磷脂转运的扩散机制。在当前的资金期间， 我们已经表明，可以简单地抑制Bamb Bame双突变体的条件致命表型 通过删除表面暴露的脂蛋白，我们揭示了存在替代性脂蛋白运输的存在 途径，我们发现了肠杆菌公共抗原的循环形式在维持OM中的作用 障碍物，我们确定了激活或启动σE应力响应的突变，以抑制多种OMP 和BAM缺陷。在翻译研究中，我们使用对OM生物发生的知识来发现一类新的 可在细胞表面抑制巴马的抗生素。 我们建议使用大量的突变，这些突变会改变BAM组件或各种OMP底物 以及我们的补充剂集合作为探索OMP组装过程的工具。特别是，我们会 探测非必需的BAMBCE脂蛋白的功能，并测试我们的假设，即BAMD不 发挥真正的基本机械作用，而是作为控制巴马活性的调节剂。我们 将测试伴侣SKP作为周质蛋白酶DEGP的特定适配器的作用。我们也定位 通过提供 多个相互作用的面，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的O-抗原连接酶和酶排序酶连接肽 或蛋白质到LPS以挑战LPTDE Clressocon的能力。我们还将测试我们的模型三个 必需的IM蛋白质，YEJM，YCIM和FTSH构成了一种调节LPS合成的新途径 达到OM的脂质状态。 MLAA*突变通过增加LPS水平破坏了OM。这会导致OM损失膜 囊泡和IM流入OM以取代损失。我们已经确定了一个减慢这种脂质的突变 流动，我们认为对该基因的持续研究可能会提供对知识不佳的过程的见解 航空运输。