Abstract
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 Shigella Enterotoxins 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.
Highlights
Enteric infections resulting in diarrhea are prevalent throughout the developed and developing world
Previous studies with enteroinvasive E. coli required iron depletion in order to detect an enterotoxin effect in the rabbit ileal loop model and increases in Isc in the Ussing chambers [21]. These iron-depleted conditions were continued with studies for S. flexneri; it was never determined if iron-depletion was required to induce enterotoxin expression in S. flexneri
Supernatants from 2457T cultures prepared with and without the iron chelator both resulted in a significant increase in Isc, and no significant difference was detected between the two conditions on rabbit tissue (Figure 1)
Summary
Enteric infections resulting in diarrhea are prevalent throughout the developed and developing world. Shigella species comprise a leading cause of diarrhea-associated morbidity and mortality in children under the age of five years in developing countries [3]. This age group is susceptible to the effects of acute and chronic infections, Shigella is clinically important in all age groups in the developing world. In addition to dehydration and the inability to absorb nutrients, children who are continuously exposed to pathogenic bacteria suffer from both physical and developmental milestone delays [1] Stools from these children frequently contain multiple infectious organisms; Shigella flexneri 2a is the most prominent bacterial pathogen isolated from children suffering from diarrhea between the ages of 2 and 5 years [3]. These toxins have diverse activities that include, but are not limited to, colonization, proteolytic activity, cellular toxicity, and increasing intestinal secretion [6,7]
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