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

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.

Figure 1

IFN-α inhibits Shigella uptake. HeLa cells were treated with 0, 50, and 500 U/ml of IFN-α, challenged with Shigella (a) or Salmonella (b) for 30 min, and the percentage of intracellular bacteria was determined by the gentamicin assay (Isberg and Falkow, 1985).

Figure 2

IFN-α affects early events in Shigella entry and inhibits foci formation. Cells (Untreated or treated with 500 U/ml of IFN-α) were challenged with Shigella expressing the AfaE adhesin for various time points (5–30 min). Shigella entry was measured using specific inside/outside stains. (a) Staining of total bacteria after permeabilization (red). (b) Staining of extracellular bacteria before cell permeabilization (blue). (c) Foci were visualized by labeling F-actin with Bodipy-phallacidin. (d) Bacteria and foci were counted automatically using dedicated computer programs (Materials and Methods), the analysis was performed for ten microscope fields per time points; bacteria labeled in a but not in b were scored as internal and represented as red spots (arrows indicate such an example); bacteria labeled with both fluorochromes were scored as external (blue spots); scored foci were represented as green squares (arrowheads indicate an example). (e) Percentage of internal/total bacteria. (f) Average number of foci per field. Filled bars, untreated cells; hatched bars, cells treated by IFN-α. Bar, 10 μm.

Figure 2

IFN-α affects early events in Shigella entry and inhibits foci formation. Cells (Untreated or treated with 500 U/ml of IFN-α) were challenged with Shigella expressing the AfaE adhesin for various time points (5–30 min). Shigella entry was measured using specific inside/outside stains. (a) Staining of total bacteria after permeabilization (red). (b) Staining of extracellular bacteria before cell permeabilization (blue). (c) Foci were visualized by labeling F-actin with Bodipy-phallacidin. (d) Bacteria and foci were counted automatically using dedicated computer programs (Materials and Methods), the analysis was performed for ten microscope fields per time points; bacteria labeled in a but not in b were scored as internal and represented as red spots (arrows indicate such an example); bacteria labeled with both fluorochromes were scored as external (blue spots); scored foci were represented as green squares (arrowheads indicate an example). (e) Percentage of internal/total bacteria. (f) Average number of foci per field. Filled bars, untreated cells; hatched bars, cells treated by IFN-α. Bar, 10 μm.

Figure 3

IFN-α inhibits Shigella-induced actin polymerization at the site of entry. Cells were challenged with Shigella expressing the AfaE adhesin, fixed after 10 min incubation at 37°C and stained for F-actin. 10 foci of entry were chosen randomly and analyzed by confocal microscopy. Cells were treated with 500 U/ml of IFN-α (bottom) or untreated (top) before Shigella challenge.

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.

Figure 5

Pp60c-src expression levels in stable transfectants. HeLa cells were transfected with a plasmid construct mediating expression of either wild-type chicken Src (srcK+) or a kinase inactive mutant of Src (srcK−) and stable clones were selected. Lysates of two representative clones were analyzed by Western blot analysis using anti-Src mAb. HeLa, parental HeLa cells; srcK+ cl3 and srcK+ cl7, HeLa cells transfected with wild-type Src, clones 3 and 7, respectively; srcK− cl4 and srcK+ cl5, HeLa cells transfected with wild-type Src, clones 4 and 5, respectively.

Figure 6

Src is responsible for Shigella-induced cortactin phosphorylation. srcK−, srcK+ and control cells (HeLa) were challenged with mxiD− or wild-type (WT) Shigella and their content in phosphotyrosyl proteins was analyzed by Western blot using anti-phosphotyrosine mAb. 5, 10, 15, 30, cells challenged with Shigella for 5, 10, 15, 30 min, respectively; D−, cells challenged with the Shigella mxiD mutant. Approximately 10 μg of protein was loaded per lane.

Figure 7

Expression of a kinase negative form of Src leads to inhibition of foci formation and of bacterial uptake. (a–c) Uninfected cells labeled for F-actin. (d–f) Cells were challenged for 15 min with wild-type Shigella. Samples were fixed and processed for immunolabeling of F-actin (green) and bacteria (red). A typical microscope field is shown for control cells (a and d), srcK+ cells (b and e) and for srcK− cells (c and f). (g) Foci of entry were scored 5, 10, 15, and 20 min after infection (squares, parental HeLa; diamonds, srcF; circles, srcK+; triangles, srcK−). (h) The percentage of internal/total bacteria after 15 min of infection was determined by inside/outside immunofluorescence staining scored automatically (filled bars, srcK+ cells; hatched bars, parental HeLa cells; empty bars, srcK− cells).

Figure 7

Expression of a kinase negative form of Src leads to inhibition of foci formation and of bacterial uptake. (a–c) Uninfected cells labeled for F-actin. (d–f) Cells were challenged for 15 min with wild-type Shigella. Samples were fixed and processed for immunolabeling of F-actin (green) and bacteria (red). A typical microscope field is shown for control cells (a and d), srcK+ cells (b and e) and for srcK− cells (c and f). (g) Foci of entry were scored 5, 10, 15, and 20 min after infection (squares, parental HeLa; diamonds, srcF; circles, srcK+; triangles, srcK−). (h) The percentage of internal/total bacteria after 15 min of infection was determined by inside/outside immunofluorescence staining scored automatically (filled bars, srcK+ cells; hatched bars, parental HeLa cells; empty bars, srcK− cells).

Figure 7

Expression of a kinase negative form of Src leads to inhibition of foci formation and of bacterial uptake. (a–c) Uninfected cells labeled for F-actin. (d–f) Cells were challenged for 15 min with wild-type Shigella. Samples were fixed and processed for immunolabeling of F-actin (green) and bacteria (red). A typical microscope field is shown for control cells (a and d), srcK+ cells (b and e) and for srcK− cells (c and f). (g) Foci of entry were scored 5, 10, 15, and 20 min after infection (squares, parental HeLa; diamonds, srcF; circles, srcK+; triangles, srcK−). (h) The percentage of internal/total bacteria after 15 min of infection was determined by inside/outside immunofluorescence staining scored automatically (filled bars, srcK+ cells; hatched bars, parental HeLa cells; empty bars, srcK− cells).

Figure 8

Effect of IFN-α on cells expressing constitutively activated Src. (a) Internalization of Shigella into HeLa, srcF18 and srcF39 cells. The three cell lines were treated with IFN-α and challenged with Shigella for 15 min. Extracellular bacteria were labeled before permeabilizing the cells, whereas total bacteria were labeled after permeabilization with a different fluorochrome. The bar chart present the ratio of internal/total bacteria as a percentage of the untreated cells (filled bars, untreated cells; hatched bars, cells treated by IFN-α). (b) Induction of PKR by IFN-α in HeLa, srcF18 and srcF39 cells. Cells were treated with IFN-α, lysed, and an equal amount of protein was submitted to Western blot analysis with anti-PKR (PKR) and anti-Src (Src) antibodies.