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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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Interaction of Unc119 with Shigella.(A) Localization of Unc119 at the site of Shigella infection. HeLa cells were infected with Shigella expressing DsRed. The cells were stained for Unc119 using an affinity-purified anti-Unc119 rabbit antibody. The middle panel shows a close-up of the site of infection as indicated by an arrow. Lower panel shows staining with a rabbit IgG control antibody. A representative image from 3 independent experiments is shown. Scale bar, 5 µm. (B) Association of Unc119 with Shigella in live cells. Cells expressing GFP-Unc119 were infected with Shigella expressing DsRed. The cells were infected, then washed after 30 min of incubation and observed under microscope. The localization of Unc119 and Shigella was monitored using time-lapse video microscopy with 30 sec intervals for 15 min (N = 3). (C) Dynamics of Unc119 and Shigella association. The percent of bacteria that were associated with Unc119 is shown. 200 infected cells were counted per experiment (N = 3, *P<0.001).
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pone-0005211-g002: Interaction of Unc119 with Shigella.(A) Localization of Unc119 at the site of Shigella infection. HeLa cells were infected with Shigella expressing DsRed. The cells were stained for Unc119 using an affinity-purified anti-Unc119 rabbit antibody. The middle panel shows a close-up of the site of infection as indicated by an arrow. Lower panel shows staining with a rabbit IgG control antibody. A representative image from 3 independent experiments is shown. Scale bar, 5 µm. (B) Association of Unc119 with Shigella in live cells. Cells expressing GFP-Unc119 were infected with Shigella expressing DsRed. The cells were infected, then washed after 30 min of incubation and observed under microscope. The localization of Unc119 and Shigella was monitored using time-lapse video microscopy with 30 sec intervals for 15 min (N = 3). (C) Dynamics of Unc119 and Shigella association. The percent of bacteria that were associated with Unc119 is shown. 200 infected cells were counted per experiment (N = 3, *P<0.001).

Mentions: In a next set of experiments HeLa cells were infected with DsRed expressing Shigella to study the localization of Unc119. Immunofluorescent staining showed a diffuse cytosolic distribution of Unc119 (Figure 2A). Unc119 was associated with vesicular structures in the cytosol and some of these vesicles were co-localized with the intracellular Shigella (Figure S1C). Next we examined the association of Unc119 with the internalized bacteria in live cells. Epifluorescence microscopy showed a time dependent association of Unc119-GFP with DsRed-expressing Shigella. Unc119-GFP remained associated with Shigella for up to 15 minutes (Figure 2B). The co-localization of Unc119-GFP with Shigella was highest at 30 minutes after the start of infection with 40% of the total internalized bacteria associated with Unc119-GFP (Figure 2C). The Unc119-Shigella association decreased to 10% by 60 minutes. We examined direct interaction of Unc119 with Shigella. Pull-down experiments with the Shigella lysate showed an interaction with 3 Shigella-derived proteins (Table S1), which may not be relevant as these proteins are not known to be secreted or expressed on the cell surface. At this time we do not know if Unc119 interacts with the type III secreted proteins of Shigella.


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

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

Interaction of Unc119 with Shigella.(A) Localization of Unc119 at the site of Shigella infection. HeLa cells were infected with Shigella expressing DsRed. The cells were stained for Unc119 using an affinity-purified anti-Unc119 rabbit antibody. The middle panel shows a close-up of the site of infection as indicated by an arrow. Lower panel shows staining with a rabbit IgG control antibody. A representative image from 3 independent experiments is shown. Scale bar, 5 µm. (B) Association of Unc119 with Shigella in live cells. Cells expressing GFP-Unc119 were infected with Shigella expressing DsRed. The cells were infected, then washed after 30 min of incubation and observed under microscope. The localization of Unc119 and Shigella was monitored using time-lapse video microscopy with 30 sec intervals for 15 min (N = 3). (C) Dynamics of Unc119 and Shigella association. The percent of bacteria that were associated with Unc119 is shown. 200 infected cells were counted per experiment (N = 3, *P<0.001).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667249&req=5

pone-0005211-g002: Interaction of Unc119 with Shigella.(A) Localization of Unc119 at the site of Shigella infection. HeLa cells were infected with Shigella expressing DsRed. The cells were stained for Unc119 using an affinity-purified anti-Unc119 rabbit antibody. The middle panel shows a close-up of the site of infection as indicated by an arrow. Lower panel shows staining with a rabbit IgG control antibody. A representative image from 3 independent experiments is shown. Scale bar, 5 µm. (B) Association of Unc119 with Shigella in live cells. Cells expressing GFP-Unc119 were infected with Shigella expressing DsRed. The cells were infected, then washed after 30 min of incubation and observed under microscope. The localization of Unc119 and Shigella was monitored using time-lapse video microscopy with 30 sec intervals for 15 min (N = 3). (C) Dynamics of Unc119 and Shigella association. The percent of bacteria that were associated with Unc119 is shown. 200 infected cells were counted per experiment (N = 3, *P<0.001).
Mentions: In a next set of experiments HeLa cells were infected with DsRed expressing Shigella to study the localization of Unc119. Immunofluorescent staining showed a diffuse cytosolic distribution of Unc119 (Figure 2A). Unc119 was associated with vesicular structures in the cytosol and some of these vesicles were co-localized with the intracellular Shigella (Figure S1C). Next we examined the association of Unc119 with the internalized bacteria in live cells. Epifluorescence microscopy showed a time dependent association of Unc119-GFP with DsRed-expressing Shigella. Unc119-GFP remained associated with Shigella for up to 15 minutes (Figure 2B). The co-localization of Unc119-GFP with Shigella was highest at 30 minutes after the start of infection with 40% of the total internalized bacteria associated with Unc119-GFP (Figure 2C). The Unc119-Shigella association decreased to 10% by 60 minutes. We examined direct interaction of Unc119 with Shigella. Pull-down experiments with the Shigella lysate showed an interaction with 3 Shigella-derived proteins (Table S1), which may not be relevant as these proteins are not known to be secreted or expressed on the cell surface. At this time we do not know if Unc119 interacts with the type III secreted proteins of Shigella.

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