Enterotoxin targeting and delivery mechanisms

说明(申请人提供)：产肠毒素大肠杆菌(ETEC)是第三世界国家重要的致泻性病原体，是一种生物防御威胁，因为军事人员很可能从野外非灭菌的水和食物来源中感染这种病原体。ETEC还对大量儿童死亡负有责任。在人类患者的活检组织中发现了来自革兰氏阴性细菌外膜的小泡。囊泡的产生并不是ETEC独有的，事实上，所有的革兰氏阴性细菌(包括其他生物治疗病原体)都会产生囊泡，但它们的特性一直很差，对囊泡成分在疾病中的作用知之甚少。来自非病原体的囊泡是相对无害的“空壳”，而毒力蛋白与来自ETEC等病原体的囊泡有关。我们先前已经发现，大多数分泌的生理活性不稳定肠毒素(LT)与ETEC囊泡有关。与细胞相比，它富含囊泡，既存在于囊泡内部，也存在于囊泡外部。小泡将毒力因子，如LT，从病原体直接转移到宿主细胞。ETEC囊泡表面的Lt介导囊泡的结合，结合导致小泡随后被肠上皮细胞内化，从而导致毒性。肠道毒素进入人体肠道细胞的临床后果是突然排出水分或虚弱的腹泻。这项研究的总体目标是表征囊泡和囊泡成分在ETEC肠毒素靶向和输送到人肠道细胞中的作用。这项建议的目的是表征纯化的ETEC囊泡的宿主细胞相互作用，了解囊泡的脂质和蛋白质成分如何影响细胞中毒，并评估囊泡水平在疾病中的影响。我们将利用细胞培养和共聚焦定位技术，以及建立的原位和体内模型来敏感和定量地表征有毒囊泡成分的作用。先前发现的导致囊泡产生过多和过少的大肠杆菌基因突变将被用于模型中，以评估囊泡产生如何与毒性相关。由这些目标产生的数据将揭示在人类疾病中发挥作用的细菌泡的关键特征。这些结果还将广泛影响对其他病原细菌产生的有毒小泡的作用的理解，这些小泡威胁到我们的防御能力。

DESCRIPTION (provided by applicant): Enterotoxigenic Escherichia coli (ETEC) is an important diarrheagenic pathogen in third world countries and is a biodefense threat since military personnel are likely to acquire this pathogen from non-sterile water and food sources in the field. ETEC is also responsible for a large number of fatalities in children. Vesicles derived from the outer membrane of Gram-negative bacteria have been detected in biopsies from human patients. Vesicle production is not unique to ETEC, in fact all gram-negative bacteria (including other biothreatening pathogens) produce vesicles, however they have been poorly characterized and little is known about the roles of vesicle components in disease. Whereas vesicles from nonpathogens are relatively harmless "empty shells", virulence proteins are associated with vesicles from pathogens such as ETEC. We have previously found that the majority of secreted physiologically active heat-labile enterotoxin (LT) is associated with ETEC vesicles. It is enriched in vesicles as compared to the cell and is present both inside and bound to the outside of the vesicle. Vesicles transfer virulence factors, such as LT, from the pathogen directly into host cells. LT on the surface of ETEC vesicles mediates vesicle binding, and binding causes subsequent internalization of vesicles by gut epithelial cells, leading to toxicity. The clinical consequence of enterotoxin entry into human intestinal cells is sudden water efflux or debilitating diarrhea. The overall objective of this research is to characterize the roles of vesicles and vesicle components in ETEC enterotoxin targeting and delivery into human intestinal cells. The aims of this proposal are to characterize the host cell interactions of purified ETEC vesicles, understand how the lipid and protein components of the vesicles affect cellular intoxication, and assess the impact of vesiculation levels in disease. We will utilize cell culture and confocal localization techniques, as well as an established in situ and in vivo models to sensitively and quantitatively characterize the roles of the toxic vesicle components. Previously identified mutations in E. coli genes that cause over- and underproduction of vesicles will be used in the models to evaluate how vesicle production correlates with toxicity. The data resulting from these aims will reveal key features of bacterial vesicles that function in human disease. These results will also broadly impact understanding the role of toxic vesicles produced by other pathogenic bacteria that threaten our defense capabilities.