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Multiple host kinases contribute to Akt activation during Salmonella infection.

Roppenser B, Kwon H, Canadien V, Xu R, Devreotes PN, Grinstein S, Brumell JH - PLoS ONE (2013)

Bottom Line: SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche.In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell.Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

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SopB-mediated Akt activation at Salmonella invasion ruffles is PI(3,4) P2/PI(3–5) P3-dependent.(A) HeLa cells were infected with wild type or ΔsopB mutant S. Typhimurium and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to Salmonella infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to Salmonella infection. Cells were infected with wild type S. Typhimurium and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt+Salmonella invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.
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pone-0071015-g001: SopB-mediated Akt activation at Salmonella invasion ruffles is PI(3,4) P2/PI(3–5) P3-dependent.(A) HeLa cells were infected with wild type or ΔsopB mutant S. Typhimurium and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to Salmonella infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to Salmonella infection. Cells were infected with wild type S. Typhimurium and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt+Salmonella invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.

Mentions: SopB is necessary for the activation of Akt during Salmonella invasion, as judged by western blotting with phospho-specific antibodies that recognize the active form of Akt, which is phosphorylated at Serine 473 (pAkt-Ser473) [12]. Cooper et al. previously showed that phosphorylated Akt is recruited to Salmonella ruffles in SopB-dependent manner [20]. Consistent with this earlier finding, we observed accumulation of pAkt-Ser473 at the actin-rich bacterial invasion site, which was visualized by phalloidin staining (Figure 1A). Such accumulation of pAkt-Ser473 at invasion sites was not observed following infection by a bacterial SopB-deficient (ΔsopB) mutant (Figure 1A). We also observed that cells transfected with plasmids expressing SopB showed significant increase in Akt activation compared to control (GFP-transfected) cells (Figure S1). Thus, SopB is both necessary and sufficient for Akt activation.


Multiple host kinases contribute to Akt activation during Salmonella infection.

Roppenser B, Kwon H, Canadien V, Xu R, Devreotes PN, Grinstein S, Brumell JH - PLoS ONE (2013)

SopB-mediated Akt activation at Salmonella invasion ruffles is PI(3,4) P2/PI(3–5) P3-dependent.(A) HeLa cells were infected with wild type or ΔsopB mutant S. Typhimurium and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to Salmonella infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to Salmonella infection. Cells were infected with wild type S. Typhimurium and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt+Salmonella invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.
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Related In: Results  -  Collection

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pone-0071015-g001: SopB-mediated Akt activation at Salmonella invasion ruffles is PI(3,4) P2/PI(3–5) P3-dependent.(A) HeLa cells were infected with wild type or ΔsopB mutant S. Typhimurium and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to Salmonella infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to Salmonella infection. Cells were infected with wild type S. Typhimurium and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to S. Typhimurium. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt+Salmonella invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.
Mentions: SopB is necessary for the activation of Akt during Salmonella invasion, as judged by western blotting with phospho-specific antibodies that recognize the active form of Akt, which is phosphorylated at Serine 473 (pAkt-Ser473) [12]. Cooper et al. previously showed that phosphorylated Akt is recruited to Salmonella ruffles in SopB-dependent manner [20]. Consistent with this earlier finding, we observed accumulation of pAkt-Ser473 at the actin-rich bacterial invasion site, which was visualized by phalloidin staining (Figure 1A). Such accumulation of pAkt-Ser473 at invasion sites was not observed following infection by a bacterial SopB-deficient (ΔsopB) mutant (Figure 1A). We also observed that cells transfected with plasmids expressing SopB showed significant increase in Akt activation compared to control (GFP-transfected) cells (Figure S1). Thus, SopB is both necessary and sufficient for Akt activation.

Bottom Line: SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche.In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell.Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology Program, Hospital for Sick Children, Toronto, Ontario, Canada.

ABSTRACT
SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

Show MeSH
Related in: MedlinePlus