Genetic Analysis of Protein Export

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)，位于膜之间的水隔室称为周质。这笔赠款的长期目标一直是利用大肠杆菌作为模型系统，从分子角度了解包膜生物发生的机制。这项提议很大程度上与OM生物发生有关。OM的所有成分，磷脂(PL)、脂多糖(LPS)、脂蛋白(LP)和2桶蛋白(OMP)都是在IM的细胞质或内小叶中合成的。我们已经确定了大多数必要的蛋白质，如果不是全部的话，这些蛋白质需要通过周质运输内毒素和OMP，并在OM中组装这些分子。为了解决这些蛋白质的功能，我们提出了几种策略，使我们能够识别损害内毒素和OMP装配机组件功能的突变。此外，我们正在开发一种结合双光子和全内反射荧光(TIRF)显微镜的技术，以探索这些组装机器的空间定位和相互作用。这些研究将提供有关这些装配机内部和之间的结构、功能和部件相互作用的重要信息。产生的机械论假说将在与哈佛大学丹·卡恩的实验室建立的、富有成效的合作中进行生化测试。我们已经发现了一种ABC转运蛋白，它与OM LP一起在从OM到IM的PL逆行运输中发挥作用。我们将讨论这个转运蛋白的功能，并试图利用它的特性来识别难以捉摸的顺行PL转运蛋白(S)的组成部分。我们对包膜应激反应的兴趣导致了两种新蛋白质的发现。其中一个似乎是所有包膜应激反应的全球调节器。其他在质量控制机制中的功能在SecY水平上运行，并显示出与真核细胞内质网应激诱导的细胞凋亡的显著相似之处。计划中的实验将解决这些蛋白质的功能以及控制它们活动的因素。与目前的观点相反，我们有强有力的证据表明，在大肠杆菌中存在暴露在表面的脂多糖。我们将讨论将这些分子定向到细胞表面的机制。耐多药的革兰氏阴性细菌日益受到关注。我们已经确定的OM生物发生所需的成分是有吸引力的药物靶点。 与公共卫生相关：革兰氏阴性细菌的外膜是一种屏障，保护这些生物免受包括抗生素在内的有毒化合物的伤害。我们已经确定了外膜中的两个蛋白质复合体，它们起到组装这个重要细胞器的功能，我们希望了解这些组装机器是如何在分子细节上工作的。这些知识将促进新抗生素的开发，以对抗这一重要类别病原体中日益严重的抗生素耐药性问题。

DESCRIPTION (provided by applicant): The cell envelope of Gram-negative bacteria contains two membranes, inner (IM) and outer (OM), and the aqueous compartment termed the periplasm that is located between the membranes. The long term goal of this grant has always been to understand the mechanisms of envelope biogenesis in molecular terms using Escherichia coli as a model system. Much of this proposal concerns OM biogenesis. All of the components of the OM, phospholipids (PL), lipopolysaccharide (LPS) lipoproteins (LP), and 2-barrel proteins (OMPs), are synthesized in the cytoplasm or the inner leaflet of the IM. We have identified most, if not all, of the essential proteins required to transport LPS and OMPs across the periplasm and assemble these molecules in the OM. To address the function of these proteins, we propose several strategies that will enable us to identify mutations that compromise the function of the components of both the LPS and the OMP assembly machines. In addition, we are developing a technique that combines Two-Photon and Total-Internal Reflection Fluorescent (TIRF) microscopy to probe the spatial localization of, and the interactions between these assembly machines. These studies will provide important information regarding the structure, function, and the component interactions within, and between, these assembly machines. Mechanistic hypotheses generated will be tested biochemically in an established, productive collaboration with the lab of Dan Kahne at Harvard. We have discovered an ABC transporter that functions with an OM LP in retrograde transport of PL from the OM to the IM. We will address the function of this transporter, and try to exploit its properties to identify components of the elusive anterograde PL transporter(s). Our interest in envelope stress responses has led to the discovery of two novel proteins. One of these appears to function as a global regulator of all the envelope stress responses. The other functions in a quality control mechanism that operates at the level of SecY and shows remarkable similarities to ER stress-induced apoptosis in eukaryotes. Experiments planned will address the functions of these proteins and what controls their activities. Contrary to current views, we have strong evidence for surface-exposed LPs in E. coli. We will address the mechanism that directs these molecules to the cell surface. Multi-drug resistant Gram-negative bacteria are a growing concern. The components that we have identified that are required for OM biogenesis are attractive drug targets. PUBLIC HEALTH RELEVANCE: The outer membrane of Gram-negative bacteria serves as a barrier to protect these organisms form toxic compounds, including antibiotics. We have identified two protein complexes in the outer membrane that function to assemble this essential cellular organelle, and we wish to understand how these assembly machines work in molecular detail. Such knowledge would facilitate the development of new antibiotics that would combat the growing problem of antibiotic resistance in this important class of pathogens.