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 革兰氏阴性菌的细胞被膜包含两层膜，内层（IM）和外层（OM）， 被称为周质的水室位于它们之间。我实验室的一个长期目标是 以大肠杆菌为模型系统，了解了被膜生物发生的机制。这 该提案涉及OM生物发生和维持细胞包膜生理的应激反应。所有 OM组分、磷脂（PL）、脂多糖（LPS）、脂蛋白和β-桶蛋白 外膜蛋白（OMPs）在IM的细胞质或内小叶中合成。我们已经鉴定出了 在OM中组装LPS（LptABCDEFG）和OMPs（BamABCDE）所需，我们提供了证据 IM和OM之间磷脂转运的扩散机制。在本供资期内， 我们已经证明，bamB和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，我们发现存在替代的脂蛋白运输 通过对这一途径的研究，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，我们确定了激活或启动σE应激反应的突变，这些突变抑制了多种OMP Bam缺陷在转化研究中，我们利用OM生物发生的知识发现了一类新的 在细胞表面抑制BamA的抗生素。 我们建议使用我们大量的突变，改变BAM组分或各种OMP底物 连同我们收集的抑制剂作为探测OMP组装过程的工具。特别是要 探索非必需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，而是作为控制BamA活性的调节剂。我们 将测试伴侣蛋白Skp作为周质蛋白酶DegP的特异性接头的作用。我们也 主要OMP的三聚体性质作为OM架构的全球组织者， 多个相互作用面，以允许蛋白质-蛋白质相互作用，这是形成蛋白质岛所必需的。 我们对LPS组装的研究将利用突变的O-抗原连接酶和分选酶来连接肽 或蛋白质转化为LPS以挑战LptDE易位子的能力。我们还将测试我们的模型， 必需的IM蛋白，YejM，YciM和FtsH包含一种调节LPS合成的新途径， 与OM的脂质状态有关。 mlaA* 突变通过增加LPS水平使OM不稳定。这会导致OM造成膜损失 囊泡化和IM PL流入OM以补充损失。我们已经发现了一种突变， 我们相信，对该基因的持续研究可能会为我们了解甚少的过程提供见解。 顺行PL运输。