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: 9922918
• 负责人: Thomas J. Silhavy
• 金额: $ 81.83万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922918
• 关键词: Affect; Antibiotics; Biogenesis; Biological Models; Cell surface; Cells; Charge; Combined Modality Therapy; Complex; Cytoplasm; Environment; Escherichia coli; Goals; Gram-Negative Bacteria; Hydrophobicity; Learning; Lipid Bilayers; Lipopolysaccharides; Lipoproteins; Maintenance; Membrane; Methods; Molecular; Molecular Chaperones; Monitor; Movement; Mutation; Nutrient; Phospholipids; Physiology; Process; Proteins; Signal Transduction; Stress; Structural defect; Surface; System; Testing; Transmembrane Domain; Work; aqueous; biological adaptation to stress; cell envelope; efflux pump; inhibitor/antagonist; insight; mutant; new therapeutic target; periplasm; public health relevance; small molecule inhibitor

 DESCRIPTION (provided by applicant): 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 transport LPS and OMPs across the periplasm and assemble these molecules in the OM. Recent work has provided functional insights into the periplasmic chaperones and the OM components of these two assembly machines, LptDE and BamABCDE. In addition, we have demonstrated that the Bam complex can export portions of the lipoprotein RcsF onto the cell surface by forming a highly interlocked complex in such a way that a short, unstructured, charged transmembrane domain of the lipoprotein is threaded through the lumen of an OMP β-barrel where it is protected from the hydrophobic membrane interior. To study OMP assembly we will test our hypothesis that the parvulin domains of the major periplasmic chaperone function in a regulatory manner. To probe Bam complex function we will identify the signals in unfolded OMPs that the Bam complex recognizes and we will find mutant substrates that will slow or stall the folding process so that intermediates can be characterized. Mutations that specifically affect formation of lipoprotein/OMP complexes will be identified and characterized. Our studies on LPS assembly will continue with a focus of how LPS molecules exit the LptD-E complex in the OM. Mutations that hinder LPS movement will be identified and these will be characterized using photocrosslinking methods that track LPS movement. With regard to envelope stress, we will test our hypothesis that the RcsF/OMP complex senses LPS structural defects directly, again using photocrosslinking probes to monitor RcsF movement within the OMP lumen. We will also continue our studies with the Mla system that removes PLs from the outer leaflet of the OM and probe the mechanisms that maintain the OM barrier when cells are starved for nutrients. The essential Bam and Lpt proteins represent attractive new drug targets. Indeed, these targets are accessible at the cell surface and thus not protected either by the OM barrier or by efflux pumps. But what is particularly intriguing is that even if they did not kill, Bam or Lpt inhibitors would disrupt the OM barrier rendering strains more sensitive to existing antibiotics and thus could be especially effective in combination therapies. The more we learn about the OM barrier and how it is made, the more rational and sophisticated our approaches to find small molecule inhibitors.

 描述(申请人提供)：革兰氏阴性细菌的细胞膜包含两层膜，内膜(IM)和外膜(OM)，以及位于它们之间的称为周质的水隔室。我的实验室的一个长期目标一直是以大肠杆菌作为模型系统来理解包膜生物发生的机制。这一建议涉及OM的生物发生和维持细胞膜生理的胁迫反应。OM的所有成分，磷脂、脂多糖、脂蛋白和β-Barrel Proteins(OMP)都是在IM的细胞质或内小叶中合成的。我们已经确定了运输内毒素和OMPS穿过周质并在OM中组装这些分子所需的必要蛋白质。最近的工作提供了对LptDE和BamABCDE这两台装配机的周质伴侣和OM组件的功能洞察。此外，我们还证明了BAM复合体可以将部分脂蛋白RCSF输出到细胞表面，方法是形成一个高度互锁的复合体，使脂蛋白的一个短的、无结构的、带电的跨膜结构域穿过一个ompβ桶的管腔，在那里它被保护不受疏水膜内部的影响。为了研究OMP组装，我们将检验我们的假设，即主要周质伴侣的小分子结构域以调节方式发挥功能。为了探索Bam复合体的功能，我们将识别Bam复合体识别的未折叠OMP中的信号，我们将找到会减缓或停止折叠过程的突变底物，以便对中间体进行表征。具体影响脂蛋白/OMP复合体形成的突变将被识别和表征。我们对内毒素组装的研究将继续，重点是内毒素分子如何离开OM中的LptD-E复合体。将识别阻碍内毒素运动的突变，并将使用跟踪内毒素运动的光交联法对这些突变进行表征。关于包膜应力，我们将检验我们的假设，即RCSF/OMP复合体直接感知内毒素结构缺陷，再次使用光交联探针监测RCSF在OMP管腔内的运动。我们还将继续我们对MLA系统的研究，该系统可以从OM的外层小叶中去除pls，并探索当细胞缺乏营养时维持OM屏障的机制。必需的Bam和LPT蛋白是极具吸引力的新药靶点。事实上，这些靶点可以在细胞表面接触到，因此不受OM屏障或外排泵的保护。但特别耐人寻味的是，即使它们没有杀死人，Bam或LPT抑制剂也会破坏对现有抗生素更敏感的OM屏障呈现菌株，因此在联合疗法中可能特别有效。我们对OM屏障以及它是如何制造的了解越多，我们寻找小分子抑制剂的方法就越合理和复杂。