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Shigella flexneri  is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that  Shigella  encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The   Sh igella   E ntero t oxins 1 and 2 (ShET1 and ShET2) have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by  S. flexneri . Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by  sen  or  ospD3 ), which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all  Shigella  proteins responsible for enterotoxin activity is vital to our understanding of  Shigella  pathogenicity and to our success in developing safe and effective vaccine candidates.

Chromosomal and Plasmid-Encoded Factors of Shigella flexneri Induce Secretogenic Activity Ex Vivo