Detection of in vivo ETEC Vesicle Production

性状（由申请方提供）：所有大肠埃希菌均从其外膜产生囊泡。非病原体产生的囊泡是相对无害的“空壳”，而病原体产生的囊泡包装有毒力蛋白。本研究的总体目标是了解病原体源性囊泡在疾病中的作用。囊泡可能是由病原体在定殖期间在宿主中产生的，然而这还没有通过实验解决。本提案描述了肠毒素E.大肠杆菌（ETEC）。在第三世界国家，ETEC是一种重要的病原体，对儿童来说可能是致命的。我们已经开发了从培养上清液中纯化和表征ETEC囊泡的方法。纯化ETEC囊泡的脂质和蛋白质组成分析指出外膜是其来源。具有生理活性的热不稳定肠毒素（LT）在ETEC囊泡中富集，并存在于囊泡的内部和外部。囊泡可以将革兰氏阴性病原体的毒力因子（如LT）直接转移到宿主细胞中。囊泡表面的LT介导肠上皮细胞对整个囊泡的结合和随后的内化。最近，我们发现了E.大肠杆菌，增加和减少水平的囊泡生产在体外，表明囊泡生产是由几个遗传位点。在这个建议中，灵敏的，定量的方法详细检测和分离体内产生的囊泡。此外，将在动物模型中评估ETEC囊泡的毒性。将体外生长的ETEC产生的纯化囊泡的毒性与体内ETEC产生的毒性进行比较。从这些目标产生的数据将回答有关病原体衍生的囊泡的可能重要的体内功能的关键问题。这些结果将为进一步研究囊泡介导的LT分泌和囊泡介导的毒力因子向宿主组织的传播奠定坚实的基础。

DESCRIPTION (provided by applicant): All Escherichia coli make vesicles derived from their outer membrane. Whereas nonpathogens produce vesicles that are relatively harmless "empty shells", pathogens produce vesicles packaged with virulence proteins. The overall objective of this research is to understand the role pathogen-derived vesicles play in disease. Vesicles are likely made in the host by pathogens during colonization, however this has not been experimentally addressed. This proposal describes the study of in vivo production of toxic vesicles by enterotoxigenic E. coli (ETEC). ETEC is an important diarrheagenic pathogen in third world countries and can be fatal for children. We have developed methods to purify and characterize ETEC vesicles from culture supernatants. Lipid and protein composition analyses of purified ETEC vesicles point to the outer membrane as their origin. Physiologically active heat-labile enterotoxin (LT) is enriched in ETEC vesicles and is present both inside and bound to the outside of the vesicle. Vesicles can transfer virulence factors, such as LT, from gram-negative pathogens directly into host cells. LT on the surface of vesicles mediates binding and subsequent internalization of entire vesicles by gut epithelial cells. Most recently, we have discovered transposon insertion mutants of E. coli that have increased and decreased levels of vesicle production in vitro, demonstrating that vesicle production is governed by several genetic loci. In this proposal, sensitive, quantitative methods are detailed to detect and isolate vesicles produced in vivo. Further, the toxicity of ETEC vesicles will be assessed in an animal model. The toxicity of purified vesicles produced by ETEC grown in vitro will be compared with those produced by ETEC in vivo. The data resulting from these aims will answer key questions regarding the likely important in vivo function of pathogen-derived vesicles. The results will provide a strong foundation for further research regarding vesicle-mediated LT secretion and vesicle-mediated dissemination of virulence factors to host tissues.