Limits...
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.

Show MeSH

Related in: MedlinePlus

Shigella triggers PKC–NF-κB activation.(A) HeLa cells were infected with Shigella. Cell lysates were prepared at the indicated time points and subjected to immunoblotting with anti-phospho-PKC antibody. *non-specific bands. (B) NF-κB luciferase assays of 293T cells transiently transfected with an NF-κB reporter plasmid and empty vector or dominant-negative forms of PKC. After 24 h, cells were infected with Shigella or treated with PMA for 3 h, and luciferase activity was measured. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (C) Cells were treated with PKCδ siRNA and transiently transfected with an NF-κB reporter plasmid. The cells were infected with Shigella and NF-κB reporter activity was measured. (D) left; HeLa cells were infected with Shigella WT, invasin expressing ΔipaB mutant, ΔvirG mutant, or Salmonella. The cell lysates were harvested at the indicated time points and subjected to immunoblot. right: A model of Shigella invasion into the epithelial cells.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675035&req=5

ppat-1003409-g003: Shigella triggers PKC–NF-κB activation.(A) HeLa cells were infected with Shigella. Cell lysates were prepared at the indicated time points and subjected to immunoblotting with anti-phospho-PKC antibody. *non-specific bands. (B) NF-κB luciferase assays of 293T cells transiently transfected with an NF-κB reporter plasmid and empty vector or dominant-negative forms of PKC. After 24 h, cells were infected with Shigella or treated with PMA for 3 h, and luciferase activity was measured. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (C) Cells were treated with PKCδ siRNA and transiently transfected with an NF-κB reporter plasmid. The cells were infected with Shigella and NF-κB reporter activity was measured. (D) left; HeLa cells were infected with Shigella WT, invasin expressing ΔipaB mutant, ΔvirG mutant, or Salmonella. The cell lysates were harvested at the indicated time points and subjected to immunoblot. right: A model of Shigella invasion into the epithelial cells.

Mentions: PMA mimics the role of diacylglycerol (DAG) in the activation of the protein kinase C (PKC)-NF-κB pathway [32]. Therefore, we hypothesized that IpaH0722 selectively targeted the DAG–PKC–NF-κB pathway, which was likely due to the membrane localization of IpaH0722 once it was secreted by invading Shigella into epithelial cells. We sought to determine which of the PKC isoforms were involved in the activation of NF-κB. The PKC family of proteins is composed of 12 isoforms that act as lipid-activated Ser/Thr kinases [33]. The PKC family can be divided into four functional protein classes: conventional PKC (PKCα, β, and γ), novel PKC (PKCδ, ε, η, and θ), atypical PKC (PKCζ, λ/ι) and PKC related kinase (PKCμ). Conventional and novel PKCs have a DAG binding domain. Conventional PKCs require DAG and Ca2+ for their activation, whereas novel PKCs require only DAG for their activation. Atypical PKCs do not depend on DAG and Ca2+ for their activation. Since the activity of PKC is regulated by phosphorylation and its recruitment to the cell membrane, we investigated the levels of phosphorylated PKC during Shigella infection of HeLa cells. HeLa cells were infected with Shigella WT and cell lysates were harvested at 10, 20, 40, and 60 min after infection for the analysis of phosphorylated PKC by immunoblotting. As shown in Fig. 3A, Shigella infection augmented the phosphorylation of conventional or novel PKCs, such as PKCδ, at 10 and 20 min, and PKCμ at 20, 40, and 60 min post-infection.


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)

Shigella triggers PKC–NF-κB activation.(A) HeLa cells were infected with Shigella. Cell lysates were prepared at the indicated time points and subjected to immunoblotting with anti-phospho-PKC antibody. *non-specific bands. (B) NF-κB luciferase assays of 293T cells transiently transfected with an NF-κB reporter plasmid and empty vector or dominant-negative forms of PKC. After 24 h, cells were infected with Shigella or treated with PMA for 3 h, and luciferase activity was measured. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (C) Cells were treated with PKCδ siRNA and transiently transfected with an NF-κB reporter plasmid. The cells were infected with Shigella and NF-κB reporter activity was measured. (D) left; HeLa cells were infected with Shigella WT, invasin expressing ΔipaB mutant, ΔvirG mutant, or Salmonella. The cell lysates were harvested at the indicated time points and subjected to immunoblot. right: A model of Shigella invasion into the epithelial cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675035&req=5

ppat-1003409-g003: Shigella triggers PKC–NF-κB activation.(A) HeLa cells were infected with Shigella. Cell lysates were prepared at the indicated time points and subjected to immunoblotting with anti-phospho-PKC antibody. *non-specific bands. (B) NF-κB luciferase assays of 293T cells transiently transfected with an NF-κB reporter plasmid and empty vector or dominant-negative forms of PKC. After 24 h, cells were infected with Shigella or treated with PMA for 3 h, and luciferase activity was measured. Results are presented as fold change relative to the activity of uninfected or unstimulated cells. *P<0.01. (C) Cells were treated with PKCδ siRNA and transiently transfected with an NF-κB reporter plasmid. The cells were infected with Shigella and NF-κB reporter activity was measured. (D) left; HeLa cells were infected with Shigella WT, invasin expressing ΔipaB mutant, ΔvirG mutant, or Salmonella. The cell lysates were harvested at the indicated time points and subjected to immunoblot. right: A model of Shigella invasion into the epithelial cells.
Mentions: PMA mimics the role of diacylglycerol (DAG) in the activation of the protein kinase C (PKC)-NF-κB pathway [32]. Therefore, we hypothesized that IpaH0722 selectively targeted the DAG–PKC–NF-κB pathway, which was likely due to the membrane localization of IpaH0722 once it was secreted by invading Shigella into epithelial cells. We sought to determine which of the PKC isoforms were involved in the activation of NF-κB. The PKC family of proteins is composed of 12 isoforms that act as lipid-activated Ser/Thr kinases [33]. The PKC family can be divided into four functional protein classes: conventional PKC (PKCα, β, and γ), novel PKC (PKCδ, ε, η, and θ), atypical PKC (PKCζ, λ/ι) and PKC related kinase (PKCμ). Conventional and novel PKCs have a DAG binding domain. Conventional PKCs require DAG and Ca2+ for their activation, whereas novel PKCs require only DAG for their activation. Atypical PKCs do not depend on DAG and Ca2+ for their activation. Since the activity of PKC is regulated by phosphorylation and its recruitment to the cell membrane, we investigated the levels of phosphorylated PKC during Shigella infection of HeLa cells. HeLa cells were infected with Shigella WT and cell lysates were harvested at 10, 20, 40, and 60 min after infection for the analysis of phosphorylated PKC by immunoblotting. As shown in Fig. 3A, Shigella infection augmented the phosphorylation of conventional or novel PKCs, such as PKCδ, at 10 and 20 min, and PKCμ at 20, 40, and 60 min post-infection.

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