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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 inhibits TRAF2-mediated NF-κB activation.(A) Luciferase reporter assays of 293T cells transiently transfected with NF-κB signaling factors 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) 293T cells were transfected with FLAG or GFP-tagged NF-κB signaling factor with or without Myc6-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP) and immunoblotting. (C) Pull down assays using GST or GST-IpaH0722 were performed with HeLa whole cell lysates. Samples were subjected to immunoblotting with anti-TRAF2 and -TRAF6.
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ppat-1003409-g005: IpaH0722 inhibits TRAF2-mediated NF-κB activation.(A) Luciferase reporter assays of 293T cells transiently transfected with NF-κB signaling factors 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) 293T cells were transfected with FLAG or GFP-tagged NF-κB signaling factor with or without Myc6-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP) and immunoblotting. (C) Pull down assays using GST or GST-IpaH0722 were performed with HeLa whole cell lysates. Samples were subjected to immunoblotting with anti-TRAF2 and -TRAF6.

Mentions: We subsequently investigated other NF-κB signaling factors, namely TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 as potential IpaH0722 targets [40]. To this end, 293T cells expressing NF-κB-luciferase reporter gene together with TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 were transfected with a vector encoding IpaH0722 or IpaH0722CA and luciferase activity was measured (Fig. 5A). IpaH0722, but not IpaH0722CA, inhibited NF-κB activity when the cells that expressed TRAF2 suggesting that TRAF2 is a target for IpaH0722 E3 ubiquitin ligase (Fig. 5A). In previous reports, PKC-mediated phosphorylation of TRAF2 was a prerequisite for NF-κB activation in epithelial cells [41], [42]. PKCδ and PKCε phosphorylated TRAF2 at residue Thr117 resulting in IKK recruitment and ultimately NF-κB activation [41], [42]. We therefore used siRNA-mediated TRAF2 knockdown cells to measure the effect of TRAF2 on activation of the PKC-NF-κB pathway. The results showed that TRAF2 knockdown decreased NF-κB activation in response to PMA stimulation and Shigella infection (Fig. S4A). To further confirm the effect of TRAF2 knockdown on NF-κB activation we utilized Traf2-knockout mouse embryonic fibroblasts (MEF) [43]. We transduced Traf2 into the Traf2-knockout MEFs by retrovirus infection to generate stably expressing Traf2 cell lines. The degradation of IκBα in Traf2 stably expressing MEF cells and Traf2-knockout MEF cells during Shigella infection was determined. As shown in Fig. S4B, the degradation rate of IκBα in Traf2 stably expressing MEFs was higher than that of Traf2-knockout MEFs at 20 and 40 min after infection suggesting that TRAF2 plays a role in Shigella-induced NF-κB activation.


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 inhibits TRAF2-mediated NF-κB activation.(A) Luciferase reporter assays of 293T cells transiently transfected with NF-κB signaling factors 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) 293T cells were transfected with FLAG or GFP-tagged NF-κB signaling factor with or without Myc6-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP) and immunoblotting. (C) Pull down assays using GST or GST-IpaH0722 were performed with HeLa whole cell lysates. Samples were subjected to immunoblotting with anti-TRAF2 and -TRAF6.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675035&req=5

ppat-1003409-g005: IpaH0722 inhibits TRAF2-mediated NF-κB activation.(A) Luciferase reporter assays of 293T cells transiently transfected with NF-κB signaling factors 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) 293T cells were transfected with FLAG or GFP-tagged NF-κB signaling factor with or without Myc6-IpaH0722CA. After 24 h, cells were harvested and subjected to immunoprecipitation (IP) and immunoblotting. (C) Pull down assays using GST or GST-IpaH0722 were performed with HeLa whole cell lysates. Samples were subjected to immunoblotting with anti-TRAF2 and -TRAF6.
Mentions: We subsequently investigated other NF-κB signaling factors, namely TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 as potential IpaH0722 targets [40]. To this end, 293T cells expressing NF-κB-luciferase reporter gene together with TRAF2, TRAF5, TRAF6, NIK, IKKα, IKKβ, or p65 were transfected with a vector encoding IpaH0722 or IpaH0722CA and luciferase activity was measured (Fig. 5A). IpaH0722, but not IpaH0722CA, inhibited NF-κB activity when the cells that expressed TRAF2 suggesting that TRAF2 is a target for IpaH0722 E3 ubiquitin ligase (Fig. 5A). In previous reports, PKC-mediated phosphorylation of TRAF2 was a prerequisite for NF-κB activation in epithelial cells [41], [42]. PKCδ and PKCε phosphorylated TRAF2 at residue Thr117 resulting in IKK recruitment and ultimately NF-κB activation [41], [42]. We therefore used siRNA-mediated TRAF2 knockdown cells to measure the effect of TRAF2 on activation of the PKC-NF-κB pathway. The results showed that TRAF2 knockdown decreased NF-κB activation in response to PMA stimulation and Shigella infection (Fig. S4A). To further confirm the effect of TRAF2 knockdown on NF-κB activation we utilized Traf2-knockout mouse embryonic fibroblasts (MEF) [43]. We transduced Traf2 into the Traf2-knockout MEFs by retrovirus infection to generate stably expressing Traf2 cell lines. The degradation of IκBα in Traf2 stably expressing MEF cells and Traf2-knockout MEF cells during Shigella infection was determined. As shown in Fig. S4B, the degradation rate of IκBα in Traf2 stably expressing MEFs was higher than that of Traf2-knockout MEFs at 20 and 40 min after infection suggesting that TRAF2 plays a role in Shigella-induced NF-κB activation.

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