Enterotoxin targeting and delivery mechanisms

描述（由申请人提供）：产肠毒素大肠杆菌（ETEC）在第三世界国家是一种重要的致腹泻病原体，是一种生物防御威胁，因为军事人员很可能从野外非无菌的水和食物来源中获得这种病原体。ETEC还造成大量儿童死亡。在人类患者的活组织检查中发现了革兰氏阴性菌外膜的囊泡。囊泡的产生并不是ETEC所独有的，事实上所有革兰氏阴性菌（包括其他生物威胁性病原体）都会产生囊泡，然而它们的特征很少，而且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”，而毒力蛋白则与来自病原体（如ETEC）的囊泡有关。我们以前发现，大多数分泌的生理活性热不稳定肠毒素（LT）与ETEC囊泡有关。与细胞相比，它在囊泡中富集，存在于囊泡内部并与囊泡外部结合。囊泡将毒力因子（如LT）从病原体直接转移到宿主细胞中。ETEC囊泡表面的LT介导囊泡结合，而这种结合导致肠上皮细胞随后将囊泡内化，从而导致毒性。肠毒素进入人体肠细胞的临床后果是突然的水外溢或虚弱的腹泻。本研究的总体目的是表征囊泡和囊泡成分在ETEC肠毒素靶向和递送到人肠细胞中的作用。本提案的目的是表征纯化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。先前确定的大肠杆菌基因突变会导致囊泡产生过多和不足，这些突变将用于模型中，以评估囊泡产生与毒性之间的关系。这些目标产生的数据将揭示在人类疾病中起作用的细菌囊泡的关键特征。这些结果也将广泛影响对其他致病细菌产生的有毒囊泡的作用的理解，这些囊泡威胁着我们的防御能力。

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.