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. ? ?