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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 Shigella-induced NF-κB activation.(A) HeLa cells were infected with Shigella WT, ΔipaH-chromosome (left), or ΔipaH0722 (right). The cell lysates were prepared at the indicated time points and subjected to immunoblottings with anti-IκBα. Anti-actin antibody was used as a loading control. The values indicated below the images are the relative intensities of the bands. (B) IpaH0722 localizes to the host cell plasma membrane. Cos-7 cells were transfected with a Myc-IpaH0722 expression vector and immunostained with anti-Myc, actin, and TO-PRO3. (C) The murine pulmonary model of Shigella infection. Mice were intranasally inoculated with Shigella WT or ΔipaH0722 (n = 13) at 5×107 cfu and survival was recorded for 10 days post-infection. *P<0.05. (D) NF-κB-luciferase and Elk-1-luciferase reporter assays were performed after Shigella infection of 293T cells that were transiently transfected with empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as ‘fold’ relative to the activity of uninfected or unstimulated cells. *P<0.01, n.s., not significant. (E) HeLa cells were infected with Shigella WT, ΔipaH0722, or ΔipaH0722 harboring ipaH0722 or ipaH0722CA. The cell lysates prepared at the indicated time points were subjected to immunoblotting. Anti-actin antibody served as the loading control.
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ppat-1003409-g001: IpaH0722 inhibits Shigella-induced NF-κB activation.(A) HeLa cells were infected with Shigella WT, ΔipaH-chromosome (left), or ΔipaH0722 (right). The cell lysates were prepared at the indicated time points and subjected to immunoblottings with anti-IκBα. Anti-actin antibody was used as a loading control. The values indicated below the images are the relative intensities of the bands. (B) IpaH0722 localizes to the host cell plasma membrane. Cos-7 cells were transfected with a Myc-IpaH0722 expression vector and immunostained with anti-Myc, actin, and TO-PRO3. (C) The murine pulmonary model of Shigella infection. Mice were intranasally inoculated with Shigella WT or ΔipaH0722 (n = 13) at 5×107 cfu and survival was recorded for 10 days post-infection. *P<0.05. (D) NF-κB-luciferase and Elk-1-luciferase reporter assays were performed after Shigella infection of 293T cells that were transiently transfected with empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as ‘fold’ relative to the activity of uninfected or unstimulated cells. *P<0.01, n.s., not significant. (E) HeLa cells were infected with Shigella WT, ΔipaH0722, or ΔipaH0722 harboring ipaH0722 or ipaH0722CA. The cell lysates prepared at the indicated time points were subjected to immunoblotting. Anti-actin antibody served as the loading control.

Mentions: The infection of mice using the ΔipaH-chromosome mutant, which lacks all of the chromosomal ipaH genes, leads to an increased production of the NF-κB responsive gene MIP-2 [28]. In this current study, we used HeLa cells to determine the effect of the ΔipaH-chromosome mutant on the NF-κB activation by measuring the kinetics of IκBα degradation. HeLa cells were infected with YSH6000 (S. flexneri WT) or ΔipaH-chromosome mutant, and then whole cell lysates were harvested at 20, 40, 60, and 80 min post-infection for the detection of IκBα. The degradation rate of IκBα in cells infected with the ΔipaH-chromosome mutant was higher than that of WT, indicating that one or more of the chromosomal IpaH proteins contributed to the dampening of IκBα degradation (Fig. 1A, left). To identify the IpaH proteins that were involved in suppressing NF-κB activation, we generated deletions mutants for each of the seven chromosomal ipaH genes. IpaH0722 was critical for the inhibition of NF-κB activation. The degradation rate of IκBα was higher in ΔipaH0722 infection compared to WT infection (Fig. 1A, right). IpaH0722, which corresponds to ORF SF0722 of Shigella flexneri 2a Sf301 strain, has a Cys residue in its C-terminal region that is required for E3 ubiquitin ligase activity [16], [28], [29].


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 Shigella-induced NF-κB activation.(A) HeLa cells were infected with Shigella WT, ΔipaH-chromosome (left), or ΔipaH0722 (right). The cell lysates were prepared at the indicated time points and subjected to immunoblottings with anti-IκBα. Anti-actin antibody was used as a loading control. The values indicated below the images are the relative intensities of the bands. (B) IpaH0722 localizes to the host cell plasma membrane. Cos-7 cells were transfected with a Myc-IpaH0722 expression vector and immunostained with anti-Myc, actin, and TO-PRO3. (C) The murine pulmonary model of Shigella infection. Mice were intranasally inoculated with Shigella WT or ΔipaH0722 (n = 13) at 5×107 cfu and survival was recorded for 10 days post-infection. *P<0.05. (D) NF-κB-luciferase and Elk-1-luciferase reporter assays were performed after Shigella infection of 293T cells that were transiently transfected with empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as ‘fold’ relative to the activity of uninfected or unstimulated cells. *P<0.01, n.s., not significant. (E) HeLa cells were infected with Shigella WT, ΔipaH0722, or ΔipaH0722 harboring ipaH0722 or ipaH0722CA. The cell lysates prepared at the indicated time points were subjected to immunoblotting. Anti-actin antibody served as the loading control.
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ppat-1003409-g001: IpaH0722 inhibits Shigella-induced NF-κB activation.(A) HeLa cells were infected with Shigella WT, ΔipaH-chromosome (left), or ΔipaH0722 (right). The cell lysates were prepared at the indicated time points and subjected to immunoblottings with anti-IκBα. Anti-actin antibody was used as a loading control. The values indicated below the images are the relative intensities of the bands. (B) IpaH0722 localizes to the host cell plasma membrane. Cos-7 cells were transfected with a Myc-IpaH0722 expression vector and immunostained with anti-Myc, actin, and TO-PRO3. (C) The murine pulmonary model of Shigella infection. Mice were intranasally inoculated with Shigella WT or ΔipaH0722 (n = 13) at 5×107 cfu and survival was recorded for 10 days post-infection. *P<0.05. (D) NF-κB-luciferase and Elk-1-luciferase reporter assays were performed after Shigella infection of 293T cells that were transiently transfected with empty vector, IpaH0722, or IpaH0722CA expressing plasmids. Results are presented as ‘fold’ relative to the activity of uninfected or unstimulated cells. *P<0.01, n.s., not significant. (E) HeLa cells were infected with Shigella WT, ΔipaH0722, or ΔipaH0722 harboring ipaH0722 or ipaH0722CA. The cell lysates prepared at the indicated time points were subjected to immunoblotting. Anti-actin antibody served as the loading control.
Mentions: The infection of mice using the ΔipaH-chromosome mutant, which lacks all of the chromosomal ipaH genes, leads to an increased production of the NF-κB responsive gene MIP-2 [28]. In this current study, we used HeLa cells to determine the effect of the ΔipaH-chromosome mutant on the NF-κB activation by measuring the kinetics of IκBα degradation. HeLa cells were infected with YSH6000 (S. flexneri WT) or ΔipaH-chromosome mutant, and then whole cell lysates were harvested at 20, 40, 60, and 80 min post-infection for the detection of IκBα. The degradation rate of IκBα in cells infected with the ΔipaH-chromosome mutant was higher than that of WT, indicating that one or more of the chromosomal IpaH proteins contributed to the dampening of IκBα degradation (Fig. 1A, left). To identify the IpaH proteins that were involved in suppressing NF-κB activation, we generated deletions mutants for each of the seven chromosomal ipaH genes. IpaH0722 was critical for the inhibition of NF-κB activation. The degradation rate of IκBα was higher in ΔipaH0722 infection compared to WT infection (Fig. 1A, right). IpaH0722, which corresponds to ORF SF0722 of Shigella flexneri 2a Sf301 strain, has a Cys residue in its C-terminal region that is required for E3 ubiquitin ligase activity [16], [28], [29].

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