Salmonella typhimurium translocates flagellin across intestinal epithelia,inducing a proinflammatory response

This study investigated whether soluble paracrine factors mediated Salmonel la-induced IL-8 expression in polarized model intestinal epithelia. We foun d that the basolateral media of model epithelia that had been apically infe cted with Salmonella typhimurium for a short period (10 minutes) could acti vate IL-8 secretion in virgin model epithelia, demonstrating that a proinfl ammatory factor (PIF) was indeed present. Initial characterization found th at PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epith elia to elicit IL-8 secretion. PIF was not present in the media of model ep ithelia stimulated with other inducers of IL-8 secretion (TNF-alpha or carb achol) but was present in S. typhimurium supernatants, indicating PIF is of bacterial origin. PIF was purified from bacterial culture supernatants by anion/cation exchange chromatography and SDS-PAGE and found by using micros equencing to be the protein flagellin. In support of this finding, flagelli n-deficient: S, typhimurium mutants did not secrete detectable levels of PI F (i.e., a bioactivity that induced IL-8 secretion when placed basolaterall y on model epithelia). Furthermore, viable flagellin-deficient mutant organ isms (fliC/fljB and flhD) failed to elicit IL-8 secretion when added apical ly to model intestinal epithelia. These findings indicate that translocatio n of flagellin across epithelia, subsequent to apical epithelial-S. typhimu rium interaction, is likely a major means of activating a mucosal. inflamma tory response.