Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells.
Bottom Line: Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells.The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells.Immunofluorescence studies showed that IFN-alpha inhibits Shigella-induced actin polymerization required for bacterial entry into cells.
Affiliation: Unité de Génétique Humaine, INSERM U276.
Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-alpha inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-alpha inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-alpha. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-alpha treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-alpha inhibits this Src-dependent signaling pathway.
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Mentions: The role of pp60c-src on the induction of actin rearrangements at the site of Shigella entry was investigated using immunofluorescence techniques. Expression of the Src constructs in HeLa cells did not result in significant changes in cell morphology (not shown). SrcK− cells (Fig. 7 c), however, presented more actin cables when compared with parental cells (Fig. 7 a). Transfectants were challenged with Shigella, and at different time points, samples were fixed and double-stained for F-actin (Fig. 7, d–f, green) and bacterial LPS (Fig. 7, d–f, red). Shigella-induced foci in srcK+ cells appeared similar to foci induced in parental cells, although moderately less abundant. In srcK− cells, however, the number of foci was drastically reduced. The average number of bacteria associated per cell was not significantly different among the different transfectants. Fig. 7 g presents the kinetics of foci formation, scored as the number of foci per cell. As expected, the number of foci increased steadily in control cells between 5 and 15 min, <0.12 foci per cell, and decreased sharply at 20 min (Fig. 7 g, squares). srcK− cells showed a marked reduction in the number of foci at all time points, peaking at 0.026 foci per cell (Fig. 7 g, triangles). Shigella uptake was then quantitated by inside/outside immunofluorescence staining. Consistent with the reduction of foci number, the amounts of internalized bacteria in the srcK− cells decreased by 75% after 15 min of infection when compared with the control cells (Fig. 7 h, empty bar). Taken together, these results indicate that pp60c-src activity is required for the formation of Shigella actin foci and for Shigella entry.