Bacterial inhibitors of eukaryotic membrane fusion

描述(由申请人提供)：嗜肺军团菌(LPN)会引起一种严重的、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名患者住院治疗。然而，由于一贯缺乏报告，这些数字可能低估了感染总人数。虽然大多数健康的人通过适当的抗生素治疗完全从感染中恢复，但老年和极年轻的患者可能会死于这种疾病，在各种疫情爆发期间，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可以入侵细胞导致疾病的机制是可取的，并可能有助于促进LPN爆发和感染的新疗法。我们的目标是研究LPN改变宿主细胞环境的机制。LPN产生和分泌许多调节正常真核过程的细菌蛋白质，我们建议重点研究那些调节真核细胞膜融合的蛋白质。LPN抑制或改变真核细胞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避宿主细胞对溶酶体降解的第一线防御。因此，识别和表征LPN改变真核膜融合和转运途径的机制将为了解LPN在细胞内存活的能力和致病能力提供新的见解。在过去的三年里，我的实验室使用了一种强大的真核膜融合的生化模型，即来自酵母的空泡的同型融合，以开始鉴定来自LPN的一种蛋白质LegC3，它现在被证明直接抑制真核膜融合。我们建议使用这个体内和体外的SCE空泡融合系统来继续研究LegC3以及来自LPN的其他3个类似的蛋白质，并将检验细胞内的病原菌，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。通过强大的膜融合模型，我们可以开始剖析LPN发病的分子机制。本申请的三个具体目的是：目的1：确认和鉴定LPNLegC3蛋白的受体(S)。目的：阐明LPN LegC3蛋白抑制真核膜融合的机制。目的：研究姐妹染色单体中另外3个LPN卷曲蛋白LegC2、LegC7和ICMG/DotF的功能。好了！

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. !