Bacterial inhibitors of eukaryotic membrane fusion

点击翻译按钮获取中文摘要

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.