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Selective inhibition of type III secretion activated signaling by the Salmonella effector AvrA.

Du F, Galán JE - PLoS Pathog. (2009)

Bottom Line: We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-kappaB activation.We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway.This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Salmonella enterica utilizes a type III secretion system (TTSS) encoded in its pathogenicity island 1 to mediate its initial interactions with intestinal epithelial cells, which are characterized by the stimulation of actin cytoskeleton reorganization and a profound reprogramming of gene expression. These responses result from the stimulation of Rho-family GTPases and downstream signaling pathways by specific effector proteins delivered by this TTSS. We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-kappaB activation. We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway. This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems.

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AvrA delivered by the TTSS inhibits the activation of the JNK but not the p38 or NF-κB pathways stimulated by S. typhimurium infection.A Transiently overexpressed AvrA inhibits TNFα-stimulated IL-8 transcription. HEK293 cells were co-transfected with a pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection), and when indicated, with a plasmid encoding AvrA, YopJ or their catalytic mutants AvrAC172S or YopJC172A. Twenty-four hours after transfection, cells were treated with TNFα (10 ng/ml) for 30 min and the stimulation of IL-8 transcription in transfected cells was measured with the Dual-Luciferase Reporter Assay System (Promega) as indicated in Materials and Methods. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. B and C Transiently overexpressed AvrA inhibits TNFα- and Salmonella infection-stimulated JNK and p38 pathways. HEK293 cells were transfected with the indicated plasmids and 24 hs after transfection, cells were either treated with TNFα (10 ng/ml) or infected with S. Typhimurium ΔavrA strain. Thirty minutes after treatment or at the indicated times after infection the activation of p38 and JNK was assayed by western immunoblot using antibodies directed to the phosphorylated (activated) form of these kinases (see Materials and Methods for details). D Transiently overexpressed AvrA inhibits TNFα-stimulated NF-κB promoter reporter transcription only very weakly. The assay was performed in the same way as in A, except that the reporter plasmid pBIIX-Luc was used, which harbors two tandem repeats of NF-κB recognition sites. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. E AvrA delivered by the S. Typhimurium TTSS inhibits the JNK signaling pathway but does not affect the p38 or NF-κB pathways. HEK293 cells were infected with S. Typhimurium strains expressing AvrA or its catalytic mutant AvrAC172S and at the indicated times after infection, the stimulation of p38 and JNK, as well as stabilization of IκBα, were assayed by western immunoblot as described in Materials and Methods. F Quantitative RT-PCR analysis of selected genes induced by S. typhimurium infection. Henle-407 intestinal epithelial cells were infected for 1 h with S. typhimurium wild type, ΔavrA or the type III secretion-defective ΔinvA strains, and then chased in DMEM supplemented with gentamicin (100 µg/ml) for 3 hs. RT-qPCR was performed as described in Materials and Methods. COX2, NFKBIA, and SOD2 are regulated by the NF-κB pathway, while JUN and FOSB are regulated by the JNK pathway. The threshold cycles for the specified genes were normalized against the reference gene GAPDH. Values represent mRNA fold induction relative to that of ΔinvA strains and are the mean±standard deviation of four independent measurements. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).
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ppat-1000595-g004: AvrA delivered by the TTSS inhibits the activation of the JNK but not the p38 or NF-κB pathways stimulated by S. typhimurium infection.A Transiently overexpressed AvrA inhibits TNFα-stimulated IL-8 transcription. HEK293 cells were co-transfected with a pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection), and when indicated, with a plasmid encoding AvrA, YopJ or their catalytic mutants AvrAC172S or YopJC172A. Twenty-four hours after transfection, cells were treated with TNFα (10 ng/ml) for 30 min and the stimulation of IL-8 transcription in transfected cells was measured with the Dual-Luciferase Reporter Assay System (Promega) as indicated in Materials and Methods. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. B and C Transiently overexpressed AvrA inhibits TNFα- and Salmonella infection-stimulated JNK and p38 pathways. HEK293 cells were transfected with the indicated plasmids and 24 hs after transfection, cells were either treated with TNFα (10 ng/ml) or infected with S. Typhimurium ΔavrA strain. Thirty minutes after treatment or at the indicated times after infection the activation of p38 and JNK was assayed by western immunoblot using antibodies directed to the phosphorylated (activated) form of these kinases (see Materials and Methods for details). D Transiently overexpressed AvrA inhibits TNFα-stimulated NF-κB promoter reporter transcription only very weakly. The assay was performed in the same way as in A, except that the reporter plasmid pBIIX-Luc was used, which harbors two tandem repeats of NF-κB recognition sites. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. E AvrA delivered by the S. Typhimurium TTSS inhibits the JNK signaling pathway but does not affect the p38 or NF-κB pathways. HEK293 cells were infected with S. Typhimurium strains expressing AvrA or its catalytic mutant AvrAC172S and at the indicated times after infection, the stimulation of p38 and JNK, as well as stabilization of IκBα, were assayed by western immunoblot as described in Materials and Methods. F Quantitative RT-PCR analysis of selected genes induced by S. typhimurium infection. Henle-407 intestinal epithelial cells were infected for 1 h with S. typhimurium wild type, ΔavrA or the type III secretion-defective ΔinvA strains, and then chased in DMEM supplemented with gentamicin (100 µg/ml) for 3 hs. RT-qPCR was performed as described in Materials and Methods. COX2, NFKBIA, and SOD2 are regulated by the NF-κB pathway, while JUN and FOSB are regulated by the JNK pathway. The threshold cycles for the specified genes were normalized against the reference gene GAPDH. Values represent mRNA fold induction relative to that of ΔinvA strains and are the mean±standard deviation of four independent measurements. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).

Mentions: Previous studies have shown that, like YopJ, transient overexpression of AvrA inhibited the NF-κB and JNK pathways [26],[27]. We were able to confirm that overexpression of AvrA in cultured mammalian cells resulted in the inhibition of TNFα-induced IL-8 promoter reporter stimulation, which is dependent NF-κB (Figure 4A), as well as TNFα-stimulated JNK and p38 phosphorylation (Figure 4B). We further demonstrated that overexpression of AvrA inhibited the JNK and p38 pathways activated by S. Typhimurium infection (Figure 4C). However, overexpression of AvrA only slightly stabilized IκBα after Salmonella infection (Figure 4C), suggesting that overexpressed AvrA may only inhibit the NF-κB pathway very weakly. Consistent with this conclusion, overexpression of AvrA resulted in a very weak inhibition of the NF-κB promoter reporter after addition of TNFα (Figure 4D).


Selective inhibition of type III secretion activated signaling by the Salmonella effector AvrA.

Du F, Galán JE - PLoS Pathog. (2009)

AvrA delivered by the TTSS inhibits the activation of the JNK but not the p38 or NF-κB pathways stimulated by S. typhimurium infection.A Transiently overexpressed AvrA inhibits TNFα-stimulated IL-8 transcription. HEK293 cells were co-transfected with a pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection), and when indicated, with a plasmid encoding AvrA, YopJ or their catalytic mutants AvrAC172S or YopJC172A. Twenty-four hours after transfection, cells were treated with TNFα (10 ng/ml) for 30 min and the stimulation of IL-8 transcription in transfected cells was measured with the Dual-Luciferase Reporter Assay System (Promega) as indicated in Materials and Methods. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. B and C Transiently overexpressed AvrA inhibits TNFα- and Salmonella infection-stimulated JNK and p38 pathways. HEK293 cells were transfected with the indicated plasmids and 24 hs after transfection, cells were either treated with TNFα (10 ng/ml) or infected with S. Typhimurium ΔavrA strain. Thirty minutes after treatment or at the indicated times after infection the activation of p38 and JNK was assayed by western immunoblot using antibodies directed to the phosphorylated (activated) form of these kinases (see Materials and Methods for details). D Transiently overexpressed AvrA inhibits TNFα-stimulated NF-κB promoter reporter transcription only very weakly. The assay was performed in the same way as in A, except that the reporter plasmid pBIIX-Luc was used, which harbors two tandem repeats of NF-κB recognition sites. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. E AvrA delivered by the S. Typhimurium TTSS inhibits the JNK signaling pathway but does not affect the p38 or NF-κB pathways. HEK293 cells were infected with S. Typhimurium strains expressing AvrA or its catalytic mutant AvrAC172S and at the indicated times after infection, the stimulation of p38 and JNK, as well as stabilization of IκBα, were assayed by western immunoblot as described in Materials and Methods. F Quantitative RT-PCR analysis of selected genes induced by S. typhimurium infection. Henle-407 intestinal epithelial cells were infected for 1 h with S. typhimurium wild type, ΔavrA or the type III secretion-defective ΔinvA strains, and then chased in DMEM supplemented with gentamicin (100 µg/ml) for 3 hs. RT-qPCR was performed as described in Materials and Methods. COX2, NFKBIA, and SOD2 are regulated by the NF-κB pathway, while JUN and FOSB are regulated by the JNK pathway. The threshold cycles for the specified genes were normalized against the reference gene GAPDH. Values represent mRNA fold induction relative to that of ΔinvA strains and are the mean±standard deviation of four independent measurements. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).
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ppat-1000595-g004: AvrA delivered by the TTSS inhibits the activation of the JNK but not the p38 or NF-κB pathways stimulated by S. typhimurium infection.A Transiently overexpressed AvrA inhibits TNFα-stimulated IL-8 transcription. HEK293 cells were co-transfected with a pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection), and when indicated, with a plasmid encoding AvrA, YopJ or their catalytic mutants AvrAC172S or YopJC172A. Twenty-four hours after transfection, cells were treated with TNFα (10 ng/ml) for 30 min and the stimulation of IL-8 transcription in transfected cells was measured with the Dual-Luciferase Reporter Assay System (Promega) as indicated in Materials and Methods. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. B and C Transiently overexpressed AvrA inhibits TNFα- and Salmonella infection-stimulated JNK and p38 pathways. HEK293 cells were transfected with the indicated plasmids and 24 hs after transfection, cells were either treated with TNFα (10 ng/ml) or infected with S. Typhimurium ΔavrA strain. Thirty minutes after treatment or at the indicated times after infection the activation of p38 and JNK was assayed by western immunoblot using antibodies directed to the phosphorylated (activated) form of these kinases (see Materials and Methods for details). D Transiently overexpressed AvrA inhibits TNFα-stimulated NF-κB promoter reporter transcription only very weakly. The assay was performed in the same way as in A, except that the reporter plasmid pBIIX-Luc was used, which harbors two tandem repeats of NF-κB recognition sites. Values represent fold induction in cells transfected with the plasmid vector alone and are the mean±standard deviation of three independent measurements. E AvrA delivered by the S. Typhimurium TTSS inhibits the JNK signaling pathway but does not affect the p38 or NF-κB pathways. HEK293 cells were infected with S. Typhimurium strains expressing AvrA or its catalytic mutant AvrAC172S and at the indicated times after infection, the stimulation of p38 and JNK, as well as stabilization of IκBα, were assayed by western immunoblot as described in Materials and Methods. F Quantitative RT-PCR analysis of selected genes induced by S. typhimurium infection. Henle-407 intestinal epithelial cells were infected for 1 h with S. typhimurium wild type, ΔavrA or the type III secretion-defective ΔinvA strains, and then chased in DMEM supplemented with gentamicin (100 µg/ml) for 3 hs. RT-qPCR was performed as described in Materials and Methods. COX2, NFKBIA, and SOD2 are regulated by the NF-κB pathway, while JUN and FOSB are regulated by the JNK pathway. The threshold cycles for the specified genes were normalized against the reference gene GAPDH. Values represent mRNA fold induction relative to that of ΔinvA strains and are the mean±standard deviation of four independent measurements. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).
Mentions: Previous studies have shown that, like YopJ, transient overexpression of AvrA inhibited the NF-κB and JNK pathways [26],[27]. We were able to confirm that overexpression of AvrA in cultured mammalian cells resulted in the inhibition of TNFα-induced IL-8 promoter reporter stimulation, which is dependent NF-κB (Figure 4A), as well as TNFα-stimulated JNK and p38 phosphorylation (Figure 4B). We further demonstrated that overexpression of AvrA inhibited the JNK and p38 pathways activated by S. Typhimurium infection (Figure 4C). However, overexpression of AvrA only slightly stabilized IκBα after Salmonella infection (Figure 4C), suggesting that overexpressed AvrA may only inhibit the NF-κB pathway very weakly. Consistent with this conclusion, overexpression of AvrA resulted in a very weak inhibition of the NF-κB promoter reporter after addition of TNFα (Figure 4D).

Bottom Line: We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-kappaB activation.We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway.This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Salmonella enterica utilizes a type III secretion system (TTSS) encoded in its pathogenicity island 1 to mediate its initial interactions with intestinal epithelial cells, which are characterized by the stimulation of actin cytoskeleton reorganization and a profound reprogramming of gene expression. These responses result from the stimulation of Rho-family GTPases and downstream signaling pathways by specific effector proteins delivered by this TTSS. We show here that AvrA, an effector protein of this TTSS, specifically inhibits the Salmonella-induced activation of the JNK pathway through its interaction with MKK7, although it does not interfere with the bacterial infection-induced NF-kappaB activation. We also show that AvrA is phosphorylated at evolutionary conserved residues by a TTSS-effector-activated ERK pathway. This interplay between effector proteins delivered by the same TTSS highlights the remarkable complexity of these systems.

Show MeSH
Related in: MedlinePlus