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: To confirm these observations and further analyze the kinetics of the Shigella entry process in cells treated with IFN-α, we measured Shigella uptake using immunofluorescence and computer-assisted analysis. To specifically analyze initial steps of the entry process, Shigella strains expressing the AfaE adhesin (Materials and Methods) were used to synchronize the infection. After allowing bacterial attachment at room temperature, samples were incubated at 37°C for different periods of time and the cells were fixed. Extracellular bacteria were labeled before permeabilizing the cells (Fig. 2 b), whereas total bacteria were labeled after permeabilization with a different fluorochrome (Fig 2 a). Images were analyzed automatically by a program that allows determination of the number of intracellular bacteria labeled with one fluorochrome (Fig. 2 d, red spots) or extracellular bacteria colabeled with both fluorochromes (Fig. 2 d, blue spots). The total number of cell-associated bacteria did not show significant variations between IFN-α–treated and untreated cells, corresponding to about three bacteria per cell. Fig. 2 e shows the ratio of internal over total bacteria for each time point. For cells that were not treated with IFN-α, the percentage of intracellular bacteria increased steadily, with values ranging from 12% at 5 min to 37% at 30 min (Fig. 2 e, filled bars, 5–30). In contrast, Shigella entered cells that were pretreated with IFN-α less efficiently, with a reduction of 50% compared with untreated cells at early time points (Fig. 2 e, hatched bars, 5–20). At the 30-min time point, however, in both treated and untreated cells, bacterial internalization was similar (∼40% of total bacteria), suggesting that expression of the AfaE adhesin masked the IFN-α inhibitory effect after prolonged incubation. Similar effects linked to the AfaE adhesin were previously observed for entry defective Shigella mutant strains (Tran Van Nhieu et al., 1997), and the use of the AfaE adhesin probably led to underestimation of the IFN inhibition. These results are consistent with data obtained in gentamicin assays and argue for an inhibitory role of IFN-α in early events during Shigella entry.