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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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Effect of phosphorylation on AvrA function.A Effect of phosphorylation on the ability of AvrA to inhibt S. Typhimurium-activated JNK activation. Henle-407 cells were infected for 30 min with a ΔavrA strain harboring a low copy plasmid expressing FLAG-epitope tagged AvrA or the indicated mutants (all expressed from the avrA native promoter), then chased in DMEM supplemented with gentamicin (100 µg/ml) for the indicated times. The activation of JNK and the phosphorylation of AvrA were monitored by western immunoblot analysis as indicated above. B Effect of AvrA phosphorylaton on its ability to inhibit S. Typhimurium-stimulated IL-8 transcription. HEK293 cells were co-transfected with pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection). Twenty four hours after transfection, cells were infected with S. Typhimurium strains expressing the indicated AvrA mutants and the stimulation of IL-8 transcription in infected 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. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).
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ppat-1000595-g006: Effect of phosphorylation on AvrA function.A Effect of phosphorylation on the ability of AvrA to inhibt S. Typhimurium-activated JNK activation. Henle-407 cells were infected for 30 min with a ΔavrA strain harboring a low copy plasmid expressing FLAG-epitope tagged AvrA or the indicated mutants (all expressed from the avrA native promoter), then chased in DMEM supplemented with gentamicin (100 µg/ml) for the indicated times. The activation of JNK and the phosphorylation of AvrA were monitored by western immunoblot analysis as indicated above. B Effect of AvrA phosphorylaton on its ability to inhibit S. Typhimurium-stimulated IL-8 transcription. HEK293 cells were co-transfected with pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection). Twenty four hours after transfection, cells were infected with S. Typhimurium strains expressing the indicated AvrA mutants and the stimulation of IL-8 transcription in infected 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. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).

Mentions: We next investigated the potential influence of AvrA phosphorylation on its function when delivered by the S. Typhimurium SPI-1 TTSS. Cultured intestinal epithelial cells were infected with S. Typhimurium strains expressing the phosphorylation site mutants AvrAS3C, AvrAS3D, AvrAS14D, or AvrAS14C from the AvrA native promoter in a low-copy plasmid, and the levels of effector protein translocation and JNK activation after infection were evaluated by western immunoblot analysis over time. Cells infected with S. Typhimurium expressing AvrAS14D showed increased levels of JNK activation in comparison to cells infected with a strain expressing wild type AvrA or AvrAS14C, despite similar levels of protein translocation (Figure 6A). AvrAS14D carries a mutation that mimics the change in charge that results from its phosphorylation at Ser14. These results indicate that introduction of a phospho mimic mutation reduces AvrA activity and therefore suggest that phosphorylation may have a negative effect on AvrA function. Cells infected with S. Typhimurium expressing AvrAS3D also showed increased level of JNK phosphorylation relative to cells infected with s strain expressing AvrAS3C or wild type (Figure 6A). However, this was probably due to reduced levels of AvrAS3D translocation (Figures 6A). In no case did the phosphorylation mutants phenocopy the catalytic mutant AvrAC172S (Figure 6A).


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

Du F, Galán JE - PLoS Pathog. (2009)

Effect of phosphorylation on AvrA function.A Effect of phosphorylation on the ability of AvrA to inhibt S. Typhimurium-activated JNK activation. Henle-407 cells were infected for 30 min with a ΔavrA strain harboring a low copy plasmid expressing FLAG-epitope tagged AvrA or the indicated mutants (all expressed from the avrA native promoter), then chased in DMEM supplemented with gentamicin (100 µg/ml) for the indicated times. The activation of JNK and the phosphorylation of AvrA were monitored by western immunoblot analysis as indicated above. B Effect of AvrA phosphorylaton on its ability to inhibit S. Typhimurium-stimulated IL-8 transcription. HEK293 cells were co-transfected with pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection). Twenty four hours after transfection, cells were infected with S. Typhimurium strains expressing the indicated AvrA mutants and the stimulation of IL-8 transcription in infected 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. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).
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ppat-1000595-g006: Effect of phosphorylation on AvrA function.A Effect of phosphorylation on the ability of AvrA to inhibt S. Typhimurium-activated JNK activation. Henle-407 cells were infected for 30 min with a ΔavrA strain harboring a low copy plasmid expressing FLAG-epitope tagged AvrA or the indicated mutants (all expressed from the avrA native promoter), then chased in DMEM supplemented with gentamicin (100 µg/ml) for the indicated times. The activation of JNK and the phosphorylation of AvrA were monitored by western immunoblot analysis as indicated above. B Effect of AvrA phosphorylaton on its ability to inhibit S. Typhimurium-stimulated IL-8 transcription. HEK293 cells were co-transfected with pIL8-luc firefly luciferase reporter plasmid along with a plasmid encoding renilla luciferase (to standardize transfection). Twenty four hours after transfection, cells were infected with S. Typhimurium strains expressing the indicated AvrA mutants and the stimulation of IL-8 transcription in infected 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. ** = P<0.001; * = P<0.05 (student t test, relative to wild type values).
Mentions: We next investigated the potential influence of AvrA phosphorylation on its function when delivered by the S. Typhimurium SPI-1 TTSS. Cultured intestinal epithelial cells were infected with S. Typhimurium strains expressing the phosphorylation site mutants AvrAS3C, AvrAS3D, AvrAS14D, or AvrAS14C from the AvrA native promoter in a low-copy plasmid, and the levels of effector protein translocation and JNK activation after infection were evaluated by western immunoblot analysis over time. Cells infected with S. Typhimurium expressing AvrAS14D showed increased levels of JNK activation in comparison to cells infected with a strain expressing wild type AvrA or AvrAS14C, despite similar levels of protein translocation (Figure 6A). AvrAS14D carries a mutation that mimics the change in charge that results from its phosphorylation at Ser14. These results indicate that introduction of a phospho mimic mutation reduces AvrA activity and therefore suggest that phosphorylation may have a negative effect on AvrA function. Cells infected with S. Typhimurium expressing AvrAS3D also showed increased level of JNK phosphorylation relative to cells infected with s strain expressing AvrAS3C or wild type (Figure 6A). However, this was probably due to reduced levels of AvrAS3D translocation (Figures 6A). In no case did the phosphorylation mutants phenocopy the catalytic mutant AvrAC172S (Figure 6A).

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