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Unc119 protects from Shigella infection by inhibiting the Abl family kinases.

Vepachedu R, Karim Z, Patel O, Goplen N, Alam R - PLoS ONE (2009)

Bottom Line: We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis.Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality.Unc119 is an inducible protein.

View Article: PubMed Central - PubMed

Affiliation: National Jewish Health, Denver, CO, USA.

ABSTRACT

Background: Bacteria engage cell surface receptors and intracellular signaling molecules to enter the cell. Unc119 is an adaptor protein, which interacts with receptors and tyrosine kinases. Its role in bacterial invasion of cells is unknown.

Methodology/principal findings: We used biochemical, molecular and cell biology approaches to identify the binding partners of Unc119, and to study the effect of Unc119 on Abl family kinases and Shigella infection. We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis. Unc119 interacts with Abl family kinases and inhibits their kinase activity. As a consequence, it inhibits Crk phosphorylation, which is essential for Shigella infection. Unc119 co-localizes with Crk and Shigella in infected cells. Shigella infectivity increases in Unc119-deficient epithelial and macrophage cells. In a mouse model of shigellosis cell-permeable TAT-Unc119 inhibits Shigella infection. Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality. Unc119 is an inducible protein. Its expression is upregulated by probacteria and bacterial products such as lipopolysacharide and sodium butyrate. The latter inhibits Shigella infection in mouse lungs but is ineffective in Unc119 deficiency.

Conclusions: Unc119 inhibits signaling pathways that are used by Shigella to enter the cell. As a consequence it provides partial but significant protection from Shigella infections. Unc119 induction in vivo boosts host defense against infections.

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Related in: MedlinePlus

Overexpression of Unc119 inhibits Shigella infection, Abl/Arg activation and Crk phosphorylation.(A) 3T3 cells were infected with the bicistronic GFP-RV retrovirus (vector) or GFP-RV-Unc119 retrovirus (Unc119). Cells were sorted for GFP expression and infected with Shigella. The results represent mean±SD of 3 independent experiments performed in triplicates (*P = 0.025). (B) Reduced Abl and Arg kinase activity ex vivo in Unc119 overexpressing cells. 3T3 cells expressing GFP-RV-Unc119 and GFP-RV were infected with Shigella and the cell lysates were collected at the noted time points. Abl and Arg kinases were immunoprecipitated and assayed for kinase activity using recombinant GST-Crk as a substrate. (C) Inhibition of Crk and CRKL phosphorylation by Unc119. Crk and CrkL were activated by Shigella and PDGF (20 ng/ml) respectively. Their tyrosine phosphorylation was examined after immunoprecipitation by western blotting using an anti-pCrk (Y221) or anti-phosphotyrosine (4G10) antibody. Equal loading was checked after reprobing the membranes with an anti-CrkII (Crk) and anti-CrkL antibody. (D) Densitometric analysis of 3 independent experiments from Figures B & C is shown. *P<0.05 compared to the same time point in the absence (-) of Unc119. (F) Reduced Crk phosphorylation in Unc119 overexpressing cells. Cells plated on a glass cover-slip were transiently transfected with GFP-RV-Unc119 and GFP-RV followed by infection with Shigella. The cells were immunostained for pCrk (red). The green cells (GFP+) indicate the expression of GFP and Unc119 on the upper panel or GFP alone on the lower panel. Scale bar, 20 µm. The right bar graph shows the number of GFP-RV-Unc119 (Unc119) and GFP-RV (vector) expressing cells that also show Crk phosphorylation. The results represent mean±SD of three independent experiments (*P<0.001).
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pone-0005211-g005: Overexpression of Unc119 inhibits Shigella infection, Abl/Arg activation and Crk phosphorylation.(A) 3T3 cells were infected with the bicistronic GFP-RV retrovirus (vector) or GFP-RV-Unc119 retrovirus (Unc119). Cells were sorted for GFP expression and infected with Shigella. The results represent mean±SD of 3 independent experiments performed in triplicates (*P = 0.025). (B) Reduced Abl and Arg kinase activity ex vivo in Unc119 overexpressing cells. 3T3 cells expressing GFP-RV-Unc119 and GFP-RV were infected with Shigella and the cell lysates were collected at the noted time points. Abl and Arg kinases were immunoprecipitated and assayed for kinase activity using recombinant GST-Crk as a substrate. (C) Inhibition of Crk and CRKL phosphorylation by Unc119. Crk and CrkL were activated by Shigella and PDGF (20 ng/ml) respectively. Their tyrosine phosphorylation was examined after immunoprecipitation by western blotting using an anti-pCrk (Y221) or anti-phosphotyrosine (4G10) antibody. Equal loading was checked after reprobing the membranes with an anti-CrkII (Crk) and anti-CrkL antibody. (D) Densitometric analysis of 3 independent experiments from Figures B & C is shown. *P<0.05 compared to the same time point in the absence (-) of Unc119. (F) Reduced Crk phosphorylation in Unc119 overexpressing cells. Cells plated on a glass cover-slip were transiently transfected with GFP-RV-Unc119 and GFP-RV followed by infection with Shigella. The cells were immunostained for pCrk (red). The green cells (GFP+) indicate the expression of GFP and Unc119 on the upper panel or GFP alone on the lower panel. Scale bar, 20 µm. The right bar graph shows the number of GFP-RV-Unc119 (Unc119) and GFP-RV (vector) expressing cells that also show Crk phosphorylation. The results represent mean±SD of three independent experiments (*P<0.001).

Mentions: Next, we examined the effect of overexpression of Unc119 on bacterial uptake. Unc119 overexpression by a bicistronic GFP-RV-Unc119 retrovirus reduced bacterial uptake by 40% (Figure 5A). Overexpression of proteins can be toxic to many cell types. To ensure that the reduced bacterial uptake was not due to the loss of cell viability, we measured LDH release from Unc119 overexpressing cells, which was similar to that from control vector-transfected cells (data not shown). Next, we studied the activation of Abl kinases and Crk phosphorylation during bacterial infection in cells overexpressing Unc119. Abl and Arg kinases were immunoprecipitated and the kinase assay was performed using GST-Crk as a substrate. In Unc119 overexpressing cells the Abl and to a lower extent, the Arg kinase activity was low compared to the cells expressing the vector alone (Figure 5B & D). This data suggest that in the presence of Unc119 Abl and Arg kinases are not fully activated by bacterial infection. The decreased Abl family kinase activation should result in decreased phosphorylation of their physiological substrates Crk and CrkL [29]. Indeed, tyrosine phosphorylation of Crk (Y221) by Shigella infection and to a lower extent, phosphorylation of CrkL by PDGF was decreased in Unc119 overexpressing cells (Figure 5C & D) at 15 and 30 min after bacterial infection. The decrease in Crk phosphorylation was also examined by immunofluorescent staining of the cells following Shigella infection. Absent or reduced Crk Y221 phosphorylation was observed in 85% cells overexpressing Unc119 as indicated by GFP co-expression (Figure 5E). This contrasted with Crk Y221 phosphorylation during infection in cells overexpressing GFP alone.


Unc119 protects from Shigella infection by inhibiting the Abl family kinases.

Vepachedu R, Karim Z, Patel O, Goplen N, Alam R - PLoS ONE (2009)

Overexpression of Unc119 inhibits Shigella infection, Abl/Arg activation and Crk phosphorylation.(A) 3T3 cells were infected with the bicistronic GFP-RV retrovirus (vector) or GFP-RV-Unc119 retrovirus (Unc119). Cells were sorted for GFP expression and infected with Shigella. The results represent mean±SD of 3 independent experiments performed in triplicates (*P = 0.025). (B) Reduced Abl and Arg kinase activity ex vivo in Unc119 overexpressing cells. 3T3 cells expressing GFP-RV-Unc119 and GFP-RV were infected with Shigella and the cell lysates were collected at the noted time points. Abl and Arg kinases were immunoprecipitated and assayed for kinase activity using recombinant GST-Crk as a substrate. (C) Inhibition of Crk and CRKL phosphorylation by Unc119. Crk and CrkL were activated by Shigella and PDGF (20 ng/ml) respectively. Their tyrosine phosphorylation was examined after immunoprecipitation by western blotting using an anti-pCrk (Y221) or anti-phosphotyrosine (4G10) antibody. Equal loading was checked after reprobing the membranes with an anti-CrkII (Crk) and anti-CrkL antibody. (D) Densitometric analysis of 3 independent experiments from Figures B & C is shown. *P<0.05 compared to the same time point in the absence (-) of Unc119. (F) Reduced Crk phosphorylation in Unc119 overexpressing cells. Cells plated on a glass cover-slip were transiently transfected with GFP-RV-Unc119 and GFP-RV followed by infection with Shigella. The cells were immunostained for pCrk (red). The green cells (GFP+) indicate the expression of GFP and Unc119 on the upper panel or GFP alone on the lower panel. Scale bar, 20 µm. The right bar graph shows the number of GFP-RV-Unc119 (Unc119) and GFP-RV (vector) expressing cells that also show Crk phosphorylation. The results represent mean±SD of three independent experiments (*P<0.001).
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Related In: Results  -  Collection

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pone-0005211-g005: Overexpression of Unc119 inhibits Shigella infection, Abl/Arg activation and Crk phosphorylation.(A) 3T3 cells were infected with the bicistronic GFP-RV retrovirus (vector) or GFP-RV-Unc119 retrovirus (Unc119). Cells were sorted for GFP expression and infected with Shigella. The results represent mean±SD of 3 independent experiments performed in triplicates (*P = 0.025). (B) Reduced Abl and Arg kinase activity ex vivo in Unc119 overexpressing cells. 3T3 cells expressing GFP-RV-Unc119 and GFP-RV were infected with Shigella and the cell lysates were collected at the noted time points. Abl and Arg kinases were immunoprecipitated and assayed for kinase activity using recombinant GST-Crk as a substrate. (C) Inhibition of Crk and CRKL phosphorylation by Unc119. Crk and CrkL were activated by Shigella and PDGF (20 ng/ml) respectively. Their tyrosine phosphorylation was examined after immunoprecipitation by western blotting using an anti-pCrk (Y221) or anti-phosphotyrosine (4G10) antibody. Equal loading was checked after reprobing the membranes with an anti-CrkII (Crk) and anti-CrkL antibody. (D) Densitometric analysis of 3 independent experiments from Figures B & C is shown. *P<0.05 compared to the same time point in the absence (-) of Unc119. (F) Reduced Crk phosphorylation in Unc119 overexpressing cells. Cells plated on a glass cover-slip were transiently transfected with GFP-RV-Unc119 and GFP-RV followed by infection with Shigella. The cells were immunostained for pCrk (red). The green cells (GFP+) indicate the expression of GFP and Unc119 on the upper panel or GFP alone on the lower panel. Scale bar, 20 µm. The right bar graph shows the number of GFP-RV-Unc119 (Unc119) and GFP-RV (vector) expressing cells that also show Crk phosphorylation. The results represent mean±SD of three independent experiments (*P<0.001).
Mentions: Next, we examined the effect of overexpression of Unc119 on bacterial uptake. Unc119 overexpression by a bicistronic GFP-RV-Unc119 retrovirus reduced bacterial uptake by 40% (Figure 5A). Overexpression of proteins can be toxic to many cell types. To ensure that the reduced bacterial uptake was not due to the loss of cell viability, we measured LDH release from Unc119 overexpressing cells, which was similar to that from control vector-transfected cells (data not shown). Next, we studied the activation of Abl kinases and Crk phosphorylation during bacterial infection in cells overexpressing Unc119. Abl and Arg kinases were immunoprecipitated and the kinase assay was performed using GST-Crk as a substrate. In Unc119 overexpressing cells the Abl and to a lower extent, the Arg kinase activity was low compared to the cells expressing the vector alone (Figure 5B & D). This data suggest that in the presence of Unc119 Abl and Arg kinases are not fully activated by bacterial infection. The decreased Abl family kinase activation should result in decreased phosphorylation of their physiological substrates Crk and CrkL [29]. Indeed, tyrosine phosphorylation of Crk (Y221) by Shigella infection and to a lower extent, phosphorylation of CrkL by PDGF was decreased in Unc119 overexpressing cells (Figure 5C & D) at 15 and 30 min after bacterial infection. The decrease in Crk phosphorylation was also examined by immunofluorescent staining of the cells following Shigella infection. Absent or reduced Crk Y221 phosphorylation was observed in 85% cells overexpressing Unc119 as indicated by GFP co-expression (Figure 5E). This contrasted with Crk Y221 phosphorylation during infection in cells overexpressing GFP alone.

Bottom Line: We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis.Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality.Unc119 is an inducible protein.

View Article: PubMed Central - PubMed

Affiliation: National Jewish Health, Denver, CO, USA.

ABSTRACT

Background: Bacteria engage cell surface receptors and intracellular signaling molecules to enter the cell. Unc119 is an adaptor protein, which interacts with receptors and tyrosine kinases. Its role in bacterial invasion of cells is unknown.

Methodology/principal findings: We used biochemical, molecular and cell biology approaches to identify the binding partners of Unc119, and to study the effect of Unc119 on Abl family kinases and Shigella infection. We employed loss-of-function and gain-in-function approaches to study the effect of Unc119 in a mouse model of pulmonary shigellosis. Unc119 interacts with Abl family kinases and inhibits their kinase activity. As a consequence, it inhibits Crk phosphorylation, which is essential for Shigella infection. Unc119 co-localizes with Crk and Shigella in infected cells. Shigella infectivity increases in Unc119-deficient epithelial and macrophage cells. In a mouse model of shigellosis cell-permeable TAT-Unc119 inhibits Shigella infection. Conversely, Unc119 knockdown in vivo results in enhanced bacterial invasion and increased lethality. Unc119 is an inducible protein. Its expression is upregulated by probacteria and bacterial products such as lipopolysacharide and sodium butyrate. The latter inhibits Shigella infection in mouse lungs but is ineffective in Unc119 deficiency.

Conclusions: Unc119 inhibits signaling pathways that are used by Shigella to enter the cell. As a consequence it provides partial but significant protection from Shigella infections. Unc119 induction in vivo boosts host defense against infections.

Show MeSH
Related in: MedlinePlus