Limits...
Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells.

Duménil G, Olivo JC, Pellegrini S, Fellous M, Sansonetti PJ, Nhieu GT - J. Cell Biol. (1998)

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.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique Humaine, INSERM U276.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Shigella induces the phosphorylation of cortactin, p40  and p260. Untreated cells (−) or cells treated with 500 U/ml of  IFN-α (+) were challenged with Shigella expressing the AfaE adhesin for various time points, lysates were analyzed by Western  blot using anti-phosphotyrosine mAb. Samples were fractionated  on gels containing 8% (a and b) and 5% (c) polyacrylamide. 0, 5,  10, 15, 20, 30, cells challenged with Shigella for 0, 5, 10, 15, 20, 30  min, respectively. WB, no bacteria; D−, cells challenged with Shigella mxiD mutant. IFN-α inhibits p260 and cortactin (cort) phosphorylation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132965&req=5

Figure 4: Shigella induces the phosphorylation of cortactin, p40 and p260. Untreated cells (−) or cells treated with 500 U/ml of IFN-α (+) were challenged with Shigella expressing the AfaE adhesin for various time points, lysates were analyzed by Western blot using anti-phosphotyrosine mAb. Samples were fractionated on gels containing 8% (a and b) and 5% (c) polyacrylamide. 0, 5, 10, 15, 20, 30, cells challenged with Shigella for 0, 5, 10, 15, 20, 30 min, respectively. WB, no bacteria; D−, cells challenged with Shigella mxiD mutant. IFN-α inhibits p260 and cortactin (cort) phosphorylation.

Mentions: Cortactin, a Src-substrate recruited at the site of Shigella entry is specifically phosphorylated on tyrosine residues (Dehio et al., 1995). Therefore, we analyzed whether phosphorylation of cortactin induced by Shigella upon entry into HeLa cells was affected by IFN-α. HeLa cells were challenged with Shigella and at different time points, cells were lysed and their content in tyrosine-phosphorylated proteins was analyzed by Western blot with anti-phosphotyrosine mAb. As previously described, phosphorylation of cortactin was induced upon Shigella entry as early as 5 min, peaking at 20 min and decreasing at 30 min (Fig. 4 a, arrowhead), with kinetics similar to the induction of F-actin foci. IFN-α pretreatment reduced overall cortactin phosphorylation (Fig. 4 a, compare + with −) and this effect was detectable at every time point, but most significantly at early time points (Fig. 4 a, 5 and 10). Also, IFN-α had an inhibitory effect on tyrosine-phosphorylation of some unidentified 68-kD species, whereas phosphorylation of other proteins was unaffected by IFN-α treatment (Fig. 4 a). Quantification of the IFN-α inhibitory effect by scanning densitometry of similar gels after detection with a fluorescent substrate (Materials and Methods) indicated that, at 5 and 10 min after infection, the levels of tyrosine-phosphorylated cortactin were reduced by ∼50% in IFN-α–treated cells.


Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells.

Duménil G, Olivo JC, Pellegrini S, Fellous M, Sansonetti PJ, Nhieu GT - J. Cell Biol. (1998)

Shigella induces the phosphorylation of cortactin, p40  and p260. Untreated cells (−) or cells treated with 500 U/ml of  IFN-α (+) were challenged with Shigella expressing the AfaE adhesin for various time points, lysates were analyzed by Western  blot using anti-phosphotyrosine mAb. Samples were fractionated  on gels containing 8% (a and b) and 5% (c) polyacrylamide. 0, 5,  10, 15, 20, 30, cells challenged with Shigella for 0, 5, 10, 15, 20, 30  min, respectively. WB, no bacteria; D−, cells challenged with Shigella mxiD mutant. IFN-α inhibits p260 and cortactin (cort) phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132965&req=5

Figure 4: Shigella induces the phosphorylation of cortactin, p40 and p260. Untreated cells (−) or cells treated with 500 U/ml of IFN-α (+) were challenged with Shigella expressing the AfaE adhesin for various time points, lysates were analyzed by Western blot using anti-phosphotyrosine mAb. Samples were fractionated on gels containing 8% (a and b) and 5% (c) polyacrylamide. 0, 5, 10, 15, 20, 30, cells challenged with Shigella for 0, 5, 10, 15, 20, 30 min, respectively. WB, no bacteria; D−, cells challenged with Shigella mxiD mutant. IFN-α inhibits p260 and cortactin (cort) phosphorylation.
Mentions: Cortactin, a Src-substrate recruited at the site of Shigella entry is specifically phosphorylated on tyrosine residues (Dehio et al., 1995). Therefore, we analyzed whether phosphorylation of cortactin induced by Shigella upon entry into HeLa cells was affected by IFN-α. HeLa cells were challenged with Shigella and at different time points, cells were lysed and their content in tyrosine-phosphorylated proteins was analyzed by Western blot with anti-phosphotyrosine mAb. As previously described, phosphorylation of cortactin was induced upon Shigella entry as early as 5 min, peaking at 20 min and decreasing at 30 min (Fig. 4 a, arrowhead), with kinetics similar to the induction of F-actin foci. IFN-α pretreatment reduced overall cortactin phosphorylation (Fig. 4 a, compare + with −) and this effect was detectable at every time point, but most significantly at early time points (Fig. 4 a, 5 and 10). Also, IFN-α had an inhibitory effect on tyrosine-phosphorylation of some unidentified 68-kD species, whereas phosphorylation of other proteins was unaffected by IFN-α treatment (Fig. 4 a). Quantification of the IFN-α inhibitory effect by scanning densitometry of similar gels after detection with a fluorescent substrate (Materials and Methods) indicated that, at 5 and 10 min after infection, the levels of tyrosine-phosphorylated cortactin were reduced by ∼50% in IFN-α–treated 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.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique Humaine, INSERM U276.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus