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Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-κB activity in invaded epithelial cells.

Ashida H, Nakano H, Sasakawa C - PLoS Pathog. (2013)

Bottom Line: Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation.These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway.IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

View Article: PubMed Central - PubMed

Affiliation: Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

ABSTRACT
NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

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IpaH0722 targets PKC–NF-κB activation but not via the CBM complex.(A) IpaH0722 inhibits PKC-induced NF-κB activation. Luciferase assays of 293T cells that were transiently transfected with dominant-active forms of PKC and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (B) HeLa cells were infected with Shigella WT or the ΔipaH0722 mutant. The cell lysates were harvested at the indicated time points and subjected to immunoblotting. (C) Cells that transiently expressed empty vector, IpaH0722, or IpaH0722CA were treated with PMA+ionomycin for the indicated time points. Cell lysates were subjected to immunoblotting. (D) Luciferase reporter assays of 293T cells were transiently transfected with NF-κB and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. (E) 293T cells were transfected with FLAG-CARMA1 with or without PKCδ-DA, Myc-IpaH0722, or Myc-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP).
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ppat-1003409-g004: IpaH0722 targets PKC–NF-κB activation but not via the CBM complex.(A) IpaH0722 inhibits PKC-induced NF-κB activation. Luciferase assays of 293T cells that were transiently transfected with dominant-active forms of PKC and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (B) HeLa cells were infected with Shigella WT or the ΔipaH0722 mutant. The cell lysates were harvested at the indicated time points and subjected to immunoblotting. (C) Cells that transiently expressed empty vector, IpaH0722, or IpaH0722CA were treated with PMA+ionomycin for the indicated time points. Cell lysates were subjected to immunoblotting. (D) Luciferase reporter assays of 293T cells were transiently transfected with NF-κB and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. (E) 293T cells were transfected with FLAG-CARMA1 with or without PKCδ-DA, Myc-IpaH0722, or Myc-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP).

Mentions: Because Shigella invasion of epithelial cells triggers the DAG–PKC–NF-κB signaling, we sought to determine the host factors in the PKC–NF-κB signaling pathway that were targeted by IpaH0722 E3 ubiquitin ligase. First, we determined which of the steps during the activation of PKC–NF-κB was targeted by IpaH0722. The NF-κB activity induced by PKC signaling factors was measured in 293T cells with ectopic expression of IpaH0722 or IpaH0722CA. The transiently transfected cells were stimulated with a subset of constitutively active PKC isoforms (PKCα, -δ, -ε, and θ) and NF-κB activity was determined. IpaH0722, but not IpaH0722CA, inhibited all PKC isoforms (PKCα, -δ, -ε, and -θ) and NF-κB activation, indicating that IpaH0722 targeted PKC itself or factors that lie directly downstream of PKC (Fig. 4A). In HeLa cells, similar levels of phosphorylated PKCδ were detected in response to Shigella WT and ΔipaH0722 infection (Fig. 4B). Moreover, no differences were detected in PKC phosphorylation in PMA plus ionomycin stimulated 293T cells expressing IpaH0722 or IpaH0722CA (Fig. 4C). In addition, IpaH0722 did not bind to PKC, and had no effect on protein stability of PKC, suggesting that IpaH0722 targets downstream of PKC rather than PKC itself (Fig. S2). We therefore focused on the CARMA-Bcl10-MALT1 (CBM) complex because the phosphorylation of CARMA1, 2, and 3 by PKC induces a conformational change that recruits Bcl10-MALT1 to CARMA (CBM signalosome), which is essential for the activation of downstream signaling [38], [39]. Therefore, we tested the effect of IpaH0722 on CBM complex formation, and found that IpaH0722 failed to inhibit CARMA1-, CARMA2-, CARMA3-, or Bcl10-induced NF-κB activity (Fig. 4D). Indeed, IpaH0722 did not bind to CARMA1-, CARMA2-, CARMA3-, or Bcl10, and had no effect on their protein stability (Fig. S3). Furthermore, since IpaH0722 did not affect PKC-mediated CARMA phosphorylation (Fig. 4E), we believe that IpaH0722 inhibited the PKC–NF-κB pathway but not via the CBM complex.


Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-κB activity in invaded epithelial cells.

Ashida H, Nakano H, Sasakawa C - PLoS Pathog. (2013)

IpaH0722 targets PKC–NF-κB activation but not via the CBM complex.(A) IpaH0722 inhibits PKC-induced NF-κB activation. Luciferase assays of 293T cells that were transiently transfected with dominant-active forms of PKC and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (B) HeLa cells were infected with Shigella WT or the ΔipaH0722 mutant. The cell lysates were harvested at the indicated time points and subjected to immunoblotting. (C) Cells that transiently expressed empty vector, IpaH0722, or IpaH0722CA were treated with PMA+ionomycin for the indicated time points. Cell lysates were subjected to immunoblotting. (D) Luciferase reporter assays of 293T cells were transiently transfected with NF-κB and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. (E) 293T cells were transfected with FLAG-CARMA1 with or without PKCδ-DA, Myc-IpaH0722, or Myc-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP).
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ppat-1003409-g004: IpaH0722 targets PKC–NF-κB activation but not via the CBM complex.(A) IpaH0722 inhibits PKC-induced NF-κB activation. Luciferase assays of 293T cells that were transiently transfected with dominant-active forms of PKC and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (B) HeLa cells were infected with Shigella WT or the ΔipaH0722 mutant. The cell lysates were harvested at the indicated time points and subjected to immunoblotting. (C) Cells that transiently expressed empty vector, IpaH0722, or IpaH0722CA were treated with PMA+ionomycin for the indicated time points. Cell lysates were subjected to immunoblotting. (D) Luciferase reporter assays of 293T cells were transiently transfected with NF-κB and empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. (E) 293T cells were transfected with FLAG-CARMA1 with or without PKCδ-DA, Myc-IpaH0722, or Myc-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP).
Mentions: Because Shigella invasion of epithelial cells triggers the DAG–PKC–NF-κB signaling, we sought to determine the host factors in the PKC–NF-κB signaling pathway that were targeted by IpaH0722 E3 ubiquitin ligase. First, we determined which of the steps during the activation of PKC–NF-κB was targeted by IpaH0722. The NF-κB activity induced by PKC signaling factors was measured in 293T cells with ectopic expression of IpaH0722 or IpaH0722CA. The transiently transfected cells were stimulated with a subset of constitutively active PKC isoforms (PKCα, -δ, -ε, and θ) and NF-κB activity was determined. IpaH0722, but not IpaH0722CA, inhibited all PKC isoforms (PKCα, -δ, -ε, and -θ) and NF-κB activation, indicating that IpaH0722 targeted PKC itself or factors that lie directly downstream of PKC (Fig. 4A). In HeLa cells, similar levels of phosphorylated PKCδ were detected in response to Shigella WT and ΔipaH0722 infection (Fig. 4B). Moreover, no differences were detected in PKC phosphorylation in PMA plus ionomycin stimulated 293T cells expressing IpaH0722 or IpaH0722CA (Fig. 4C). In addition, IpaH0722 did not bind to PKC, and had no effect on protein stability of PKC, suggesting that IpaH0722 targets downstream of PKC rather than PKC itself (Fig. S2). We therefore focused on the CARMA-Bcl10-MALT1 (CBM) complex because the phosphorylation of CARMA1, 2, and 3 by PKC induces a conformational change that recruits Bcl10-MALT1 to CARMA (CBM signalosome), which is essential for the activation of downstream signaling [38], [39]. Therefore, we tested the effect of IpaH0722 on CBM complex formation, and found that IpaH0722 failed to inhibit CARMA1-, CARMA2-, CARMA3-, or Bcl10-induced NF-κB activity (Fig. 4D). Indeed, IpaH0722 did not bind to CARMA1-, CARMA2-, CARMA3-, or Bcl10, and had no effect on their protein stability (Fig. S3). Furthermore, since IpaH0722 did not affect PKC-mediated CARMA phosphorylation (Fig. 4E), we believe that IpaH0722 inhibited the PKC–NF-κB pathway but not via the CBM complex.

Bottom Line: Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation.These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway.IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

View Article: PubMed Central - PubMed

Affiliation: Division of Bacterial Infection Biology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

ABSTRACT
NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

Show MeSH
Related in: MedlinePlus