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

 描述（由申请人提供）：革兰氏阴性菌的细胞被膜包含两层膜，内层（IM）和外层（OM），以及位于它们之间的称为周质的水室。我实验室的一个长期目标一直是以大肠杆菌为模型系统，了解包膜生物发生的机制。这一提议涉及OM生物发生和维持细胞包膜生理的应激反应。OM的所有组分、磷脂（PL）、脂多糖（LPS）、脂蛋白和β桶蛋白（OMP）均在IM的细胞质或内小叶中合成。我们已经确定了必需的蛋白质运输脂多糖和外膜蛋白跨周质和组装这些分子在OM。最近的工作提供了功能的见解，周质分子伴侣和OM组件的这两个组装机，LptDE和BamABCDE。此外，我们已经证明，Bam复合物可以通过形成高度互锁的复合物将部分脂蛋白RcsF输出到细胞表面上，以这种方式，脂蛋白的短的、非结构化的、带电荷的跨膜结构域穿过OMP β-桶的内腔，在那里它被保护免受疏水膜内部的影响。为了研究OMP组装，我们将测试我们的假设，即主要的周质伴侣的小蛋白结构域的监管方式的功能。为了探测Bam复合物功能，我们将鉴定Bam复合物识别的未折叠OMP中的信号，并且我们将发现将减缓或停止折叠过程的突变底物，以便可以表征中间体。将鉴定和表征特异性影响脂蛋白/OMP复合物形成的突变。我们对LPS组装的研究将继续关注LPS分子如何退出OM中的LptD-E复合物。将鉴定阻碍LPS移动的突变，并使用追踪LPS移动的光交联方法表征这些突变。关于包膜应力，我们将测试我们的假设，即RcsF/OMP复合物直接感知LPS结构缺陷，再次使用光交联探针监测RcsF在OMP腔内的运动。我们还将继续研究Mla系统，该系统从OM的外小叶去除PL，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蛋白代表了有吸引力的新药物靶点。事实上，这些靶标在细胞表面是可接近的，因此不受OM屏障或外排泵的保护。但特别有趣的是，即使它们没有杀死，Bam或Lpt抑制剂也会破坏OM屏障，使菌株对现有抗生素更敏感，因此在联合治疗中可能特别有效。我们对OM屏障及其形成过程了解得越多，我们寻找小分子抑制剂的方法就越合理和复杂。

 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.