Bacterial inhibitors of eukaryotic membrane fusion

描述（由申请人提供）：嗜肺军团菌（Lpn）引起一种严重的，有时是致命的肺炎，称为军团病（LD）。据估计，美国每年有多达50 000人感染LD，其中多达18 000人住院。然而，由于一直缺乏报告，这些数字可能低估了感染总数。虽然大多数健康人通过适当的抗生素治疗完全从感染中恢复过来，但老年人和极其年轻的患者可能死于这种疾病，在各种疫情期间，高达30%的住院患者死于这种呼吸道病原体。因此，更深入地了解Lpn入侵细胞导致疾病的机制是可取的，并有助于促进Lpn爆发和感染的新治疗。我们的目标是研究Lpn改变宿主细胞环境的机制。Lpn产生和分泌一些调节真核生物正常过程的细菌蛋白，我们建议重点研究那些调节真核生物细胞膜融合的蛋白。Lpn抑制或改变真核胞内膜融合途径的能力是其致病能力的关键组成部分，从而使这种微生物能够逃脱宿主细胞对溶酶体降解的前线防御。因此，识别和表征Lpn改变真核膜融合和运输途径的机制将为Lpn在细胞内存活的能力及其致病能力提供新的见解。在过去的3年里，我的实验室采用了一种强大的真核膜融合生化模型，即来自酿酒酵母（Saccharomyces cerevisiae, Sce）的液泡的同型融合，开始对Lpn中的一种蛋白质LegC3进行表征，该蛋白质现在被证明可以直接抑制真核膜融合。我们建议使用这种体内和体外Sce液泡融合系统继续研究LegC3，以及来自Lpn的其他3种类似蛋白，并将验证细胞内致病菌，如Lpn，可以通过极其保守的真核核心融合机制直接改变膜融合事件的假设。通过使用强大的膜融合模型，我们可以开始剖析Lpn发病的分子机制。该应用程序的三个具体目标是：目标1：确认和表征Lpn LegC3蛋白的受体。目的2：阐明Lpn LegC3蛋白抑制真核细胞膜融合的机制。目的3：探索来自Sce的另外三种Lpn卷曲蛋白LegC2， LegC7和IcmG/DotF的功能。！

DESCRIPTION (provided by applicant): Legionella pneumophila (Lpn) causes a severe, sometimes fatal, form of pneumonia known as Legionnaires' disease (LD). It is estimated that up to 50,000 individuals in the United States contract LD every year, with up to 18,000 of these patients being hospitalized. These numbers likely underestimate the total number of infections, however, due to a consistent lack of reporting. While most healthy individuals recover completely from their infections with appropriate antibiotic treatment, elderly and extremely young patients can succumb to this disease, with up to 30% of hospitalized patients succumbing to this respiratory pathogen during various outbreaks. Therefore, a deeper understanding of the mechanisms by which Lpn can invade cells to cause disease is desirable, and could help promote new treatments for Lpn outbreaks and infections. Our goals are to study the mechanisms through which Lpn alters its host cell environment. Lpn produces and secretes a number of bacterial proteins that modulate normal eukaryotic processes, and we propose focusing on those proteins which modulate eukaryotic intracellular membrane fusion. Lpn's ability to inhibit or alter eukaryotic intracellular membrane fusion pathways is a critical component of its pathogenic capacity, thereby enabling this microorganism to escape the host cell's front-line defense of lysosomal degradation. Therefore, identifying and characterizing the mechanisms by which Lpn alters eukaryotic membrane fusion and trafficking pathways will provide new insights into Lpn's ability to survive intracellularly, and into its disease-causing capabilities. Over the past 3 years, my laboratory has employed a powerful biochemical model of eukaryotic membrane fusion, the homotypic fusion of vacuoles from the yeast Saccharomyces cerevisiae (Sce), to begin the characterization of a protein from Lpn, LegC3, that is now shown to directly inhibit eukaryotic membrane fusion. We propose using this in vivo and in vitro Sce vacuole fusion system to continue studying LegC3, as well as 3 other similar proteins from Lpn, and will test the hypothesis that intracellular pathogenic bacteria, such as Lpn, can directly alter membrane fusion events through extremely conserved, eukaryotic core fusion machinery. By using powerful models of membrane fusion, we can begin to dissect the molecular mechanisms of Lpn pathogenesis. The three specific aims of this application are: Aim 1: Confirm and characterize the receptor(s) for the Lpn LegC3 protein. Aim 2: Elucidate the mechanism by which the Lpn LegC3 protein inhibits eukaryotic membrane fusion. Aim 3: Explore the function of the three additional Lpn coiled-coil proteins LegC2, LegC7, and IcmG/DotF from Sce. !