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The Shigella type three secretion system effector OspG directly and specifically binds to host ubiquitin for activation.

Zhou Y, Dong N, Hu L, Shao F - PLoS ONE (2013)

Bottom Line: OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase.GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding.Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, China.

ABSTRACT
The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.

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Ubiquitin binding of OspG is required for inhibiting host NF-κB signaling during Shigella infection.(A) The role of OspG in recruitment of ubiquitin to intracellular Shigella. HeLa cells were infected with wild type (WT) or ΔospG mutant S. flexneri strain. Polyubiquitinated proteins stained by the FK1 antibody (green), intracellular bacteria stained by anti-Shigella antibody (red) and DAPI-stained nuclei (blue) were visualized by fluorescence microscopy. (B) The role of OspG ubiquitin binding activity in inhibiting NF-κB activation in Shigella-infected cells. HeLa cells were infected with indicated S. flexneri strains. Infected cells were treated with TNFα to stimulate NF-κB pathway activation. Lysates of infected cell collected at indicated time points after the stimulation were subjected to anti-IκBα and anti-tubulin immunoblotting analyses.
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pone-0057558-g006: Ubiquitin binding of OspG is required for inhibiting host NF-κB signaling during Shigella infection.(A) The role of OspG in recruitment of ubiquitin to intracellular Shigella. HeLa cells were infected with wild type (WT) or ΔospG mutant S. flexneri strain. Polyubiquitinated proteins stained by the FK1 antibody (green), intracellular bacteria stained by anti-Shigella antibody (red) and DAPI-stained nuclei (blue) were visualized by fluorescence microscopy. (B) The role of OspG ubiquitin binding activity in inhibiting NF-κB activation in Shigella-infected cells. HeLa cells were infected with indicated S. flexneri strains. Infected cells were treated with TNFα to stimulate NF-κB pathway activation. Lysates of infected cell collected at indicated time points after the stimulation were subjected to anti-IκBα and anti-tubulin immunoblotting analyses.

Mentions: Ubiquitin or ubiquitin-chain decoration of intracellular bacteria has been proposed as a signal that triggers autophagy-mediated host defense. To probe the functional consequence of OspG ubiquitin binding and activation of its kinase activity during Shigella infection, we first checked whether OspG plays a role in ubiquitin-chain formation surrounding intracellular Shigella. Using an antibody specific for polyubiquitinated proteins (FK1), we were able to detect some polyubiquitin signals around a portion of intracellular S. flexneri in infected HeLa cells, but this effect was not affected by deletion of ospG from the bacteria (Fig. 6A). This suggests that OspG does not play a role in recruitment or generation of polyubiquitinated proteins around S. flexneri during infection. Previous study suggests that OspG may interfere with the innate immune NF-κB signaling in S. flexneri-infected host cells [24]. Confirming this observation, TNFα treatment induced a more rapid degradation of IκBα inhibitory protein in HeLa cells infected with the ΔospG strain compared with that in wild-type strain (Fig. 6B). While re-expression of wild-type OspG in the deletion strain restored the inhibition of TNFα-induced IκBα degradation, the ubiquitin binding-deficient L190D/L191D mutant of OspG failed to complement the deletion strain in blocking IκBα degradation (Fig. 6B). These results suggest that OspG requires binding to host ubiquitin to exert its function in suppressing host innate immune signaling, which is likely through activation of its kinase activity.


The Shigella type three secretion system effector OspG directly and specifically binds to host ubiquitin for activation.

Zhou Y, Dong N, Hu L, Shao F - PLoS ONE (2013)

Ubiquitin binding of OspG is required for inhibiting host NF-κB signaling during Shigella infection.(A) The role of OspG in recruitment of ubiquitin to intracellular Shigella. HeLa cells were infected with wild type (WT) or ΔospG mutant S. flexneri strain. Polyubiquitinated proteins stained by the FK1 antibody (green), intracellular bacteria stained by anti-Shigella antibody (red) and DAPI-stained nuclei (blue) were visualized by fluorescence microscopy. (B) The role of OspG ubiquitin binding activity in inhibiting NF-κB activation in Shigella-infected cells. HeLa cells were infected with indicated S. flexneri strains. Infected cells were treated with TNFα to stimulate NF-κB pathway activation. Lysates of infected cell collected at indicated time points after the stimulation were subjected to anti-IκBα and anti-tubulin immunoblotting analyses.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585378&req=5

pone-0057558-g006: Ubiquitin binding of OspG is required for inhibiting host NF-κB signaling during Shigella infection.(A) The role of OspG in recruitment of ubiquitin to intracellular Shigella. HeLa cells were infected with wild type (WT) or ΔospG mutant S. flexneri strain. Polyubiquitinated proteins stained by the FK1 antibody (green), intracellular bacteria stained by anti-Shigella antibody (red) and DAPI-stained nuclei (blue) were visualized by fluorescence microscopy. (B) The role of OspG ubiquitin binding activity in inhibiting NF-κB activation in Shigella-infected cells. HeLa cells were infected with indicated S. flexneri strains. Infected cells were treated with TNFα to stimulate NF-κB pathway activation. Lysates of infected cell collected at indicated time points after the stimulation were subjected to anti-IκBα and anti-tubulin immunoblotting analyses.
Mentions: Ubiquitin or ubiquitin-chain decoration of intracellular bacteria has been proposed as a signal that triggers autophagy-mediated host defense. To probe the functional consequence of OspG ubiquitin binding and activation of its kinase activity during Shigella infection, we first checked whether OspG plays a role in ubiquitin-chain formation surrounding intracellular Shigella. Using an antibody specific for polyubiquitinated proteins (FK1), we were able to detect some polyubiquitin signals around a portion of intracellular S. flexneri in infected HeLa cells, but this effect was not affected by deletion of ospG from the bacteria (Fig. 6A). This suggests that OspG does not play a role in recruitment or generation of polyubiquitinated proteins around S. flexneri during infection. Previous study suggests that OspG may interfere with the innate immune NF-κB signaling in S. flexneri-infected host cells [24]. Confirming this observation, TNFα treatment induced a more rapid degradation of IκBα inhibitory protein in HeLa cells infected with the ΔospG strain compared with that in wild-type strain (Fig. 6B). While re-expression of wild-type OspG in the deletion strain restored the inhibition of TNFα-induced IκBα degradation, the ubiquitin binding-deficient L190D/L191D mutant of OspG failed to complement the deletion strain in blocking IκBα degradation (Fig. 6B). These results suggest that OspG requires binding to host ubiquitin to exert its function in suppressing host innate immune signaling, which is likely through activation of its kinase activity.

Bottom Line: OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase.GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding.Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, Beijing Normal University, Beijing, China.

ABSTRACT
The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells.

Show MeSH
Related in: MedlinePlus