Detection of in vivo ETEC Vesicle Production

描述（由申请人提供）：所有大肠杆菌的外膜产生囊泡。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裹着毒力蛋白。本研究的总体目标是了解病原体来源的囊泡在疾病中的作用。在定植过程中，病原体可能在宿主体内产生囊泡，但这尚未得到实验解决。本文描述了产肠毒素大肠杆菌（ETEC）在体内产生有毒囊泡的研究。ETEC在第三世界国家是一种重要的致腹泻病原体，对儿童可能是致命的。我们开发了从培养上清液中纯化和表征ETEC囊泡的方法。脂质和蛋白质组成分析纯化的ETEC囊泡指向外膜作为其起源。生理性活性热不稳定肠毒素（LT）在ETEC囊泡中富集，存在于囊泡内部和外部。囊泡可以将毒力因子（如LT）从革兰氏阴性病原体直接转移到宿主细胞中。囊泡表面的LT介导整个囊泡被肠上皮细胞结合并随后内化。最近，我们发现大肠杆菌的转座子插入突变体在体外可增加或减少囊泡的产生，这表明囊泡的产生是由几个基因位点控制的。在本建议中，详细介绍了灵敏的定量方法来检测和分离体内产生的囊泡。此外，ETEC囊泡的毒性将在动物模型中进行评估。将体外培养的ETEC产生的纯化囊泡的毒性与体内培养的ETEC产生的毒性进行比较。这些目标所产生的数据将回答关于病原体衍生囊泡可能重要的体内功能的关键问题。该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提供坚实的基础。

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.