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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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The carboxyl terminus of OspG is required for interaction with ubiquitin.(A) Schematic presentation of OspG truncations assayed in (B-D). The gray box donates the region that shows sequence similarity to eukaryotic serine/threonine kinase sub-domains I-VII. (B, C and D) Pulldown assays of the binding between ubiquitin and various OspG truncation proteins. Ni-NTA Sepharose beads coated with His6-ubiquitin were incubated with GST, GST-OspG or indicated GST-OspG truncation proteins. Proteins retained on the beads were eluted and then subjected to SDS-PAGE and Coomassie blue staining analysis. (E) Coimmunoprecipitation assay of OspG (WT or L190D/L191D mutant) and ubiquitin interaction in transfected HEK 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input). (F) Assay of OspG and ubiquitin interaction during Shigella infection. HEK 293T cells were infected with indicated Shigella deletion/complementation strains. Lysates of infected cell were subjected to anti-Flag immunoprecipitation. ΔmxiH is a type III secretion deficient strain; pME6032 is a rescue plasmid for expressing Flag-OspG (wild-type or L190D/L191D mutant) in the bacteria. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).
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pone-0057558-g003: The carboxyl terminus of OspG is required for interaction with ubiquitin.(A) Schematic presentation of OspG truncations assayed in (B-D). The gray box donates the region that shows sequence similarity to eukaryotic serine/threonine kinase sub-domains I-VII. (B, C and D) Pulldown assays of the binding between ubiquitin and various OspG truncation proteins. Ni-NTA Sepharose beads coated with His6-ubiquitin were incubated with GST, GST-OspG or indicated GST-OspG truncation proteins. Proteins retained on the beads were eluted and then subjected to SDS-PAGE and Coomassie blue staining analysis. (E) Coimmunoprecipitation assay of OspG (WT or L190D/L191D mutant) and ubiquitin interaction in transfected HEK 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input). (F) Assay of OspG and ubiquitin interaction during Shigella infection. HEK 293T cells were infected with indicated Shigella deletion/complementation strains. Lysates of infected cell were subjected to anti-Flag immunoprecipitation. ΔmxiH is a type III secretion deficient strain; pME6032 is a rescue plasmid for expressing Flag-OspG (wild-type or L190D/L191D mutant) in the bacteria. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).

Mentions: OspG is a small-size protein with only 196 amino acids and its sequence from residue 24 to 160 exhibits confident similarities to sub-domains I to VII of eukaryotic serine/threonine kinase (Fig. 3A and Fig. 4). The extreme carboxyl terminus (residues 161–196), predicted to form an α helix, does not show any sequence similarity to known proteins. The fact that no classical ubiquitin-binding domains or motifs could be identified in OspG indicates a novel ubiquitin-binding mode and also promoted us to further investigate its ubiquitin binding mechanism. Truncation of the amino terminal 21 residues preceding the putative kinase domain did not affect OspG binding to ubiquitin in the in vitro pulldown assay (Fig. 3B). Notably, removal of the extreme carboxyl terminal 26 residues in OspG completely abolished its interaction with ubiquitin (Fig. 3C), suggesting that the carboxyl terminal region following the kinase-like domain in OspG is essential for ubiquitin binding. Mutation of two leucine residues (L190D/L191D) in this region abolishedOspG binding to ubiquitin (Fig. 3C), suggesting the importance of these two residues. Furthermore, when the carboxyl terminal 26, 31, or 35 residues were artificially fused to GST protein and the fusion proteins were assayed for co-precipitation by ubiquitin, none of the three chimeric proteins were capable of binding to ubiquitin (Fig. 3D). These data suggest that the carboxyl terminal helical region in OspG is essential but not sufficient for mediating specific interaction with ubiquitin and the kinase-like domain is also involved in ubiquitin binding.


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)

The carboxyl terminus of OspG is required for interaction with ubiquitin.(A) Schematic presentation of OspG truncations assayed in (B-D). The gray box donates the region that shows sequence similarity to eukaryotic serine/threonine kinase sub-domains I-VII. (B, C and D) Pulldown assays of the binding between ubiquitin and various OspG truncation proteins. Ni-NTA Sepharose beads coated with His6-ubiquitin were incubated with GST, GST-OspG or indicated GST-OspG truncation proteins. Proteins retained on the beads were eluted and then subjected to SDS-PAGE and Coomassie blue staining analysis. (E) Coimmunoprecipitation assay of OspG (WT or L190D/L191D mutant) and ubiquitin interaction in transfected HEK 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input). (F) Assay of OspG and ubiquitin interaction during Shigella infection. HEK 293T cells were infected with indicated Shigella deletion/complementation strains. Lysates of infected cell were subjected to anti-Flag immunoprecipitation. ΔmxiH is a type III secretion deficient strain; pME6032 is a rescue plasmid for expressing Flag-OspG (wild-type or L190D/L191D mutant) in the bacteria. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).
© Copyright Policy
Related In: Results  -  Collection

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pone-0057558-g003: The carboxyl terminus of OspG is required for interaction with ubiquitin.(A) Schematic presentation of OspG truncations assayed in (B-D). The gray box donates the region that shows sequence similarity to eukaryotic serine/threonine kinase sub-domains I-VII. (B, C and D) Pulldown assays of the binding between ubiquitin and various OspG truncation proteins. Ni-NTA Sepharose beads coated with His6-ubiquitin were incubated with GST, GST-OspG or indicated GST-OspG truncation proteins. Proteins retained on the beads were eluted and then subjected to SDS-PAGE and Coomassie blue staining analysis. (E) Coimmunoprecipitation assay of OspG (WT or L190D/L191D mutant) and ubiquitin interaction in transfected HEK 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input). (F) Assay of OspG and ubiquitin interaction during Shigella infection. HEK 293T cells were infected with indicated Shigella deletion/complementation strains. Lysates of infected cell were subjected to anti-Flag immunoprecipitation. ΔmxiH is a type III secretion deficient strain; pME6032 is a rescue plasmid for expressing Flag-OspG (wild-type or L190D/L191D mutant) in the bacteria. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).
Mentions: OspG is a small-size protein with only 196 amino acids and its sequence from residue 24 to 160 exhibits confident similarities to sub-domains I to VII of eukaryotic serine/threonine kinase (Fig. 3A and Fig. 4). The extreme carboxyl terminus (residues 161–196), predicted to form an α helix, does not show any sequence similarity to known proteins. The fact that no classical ubiquitin-binding domains or motifs could be identified in OspG indicates a novel ubiquitin-binding mode and also promoted us to further investigate its ubiquitin binding mechanism. Truncation of the amino terminal 21 residues preceding the putative kinase domain did not affect OspG binding to ubiquitin in the in vitro pulldown assay (Fig. 3B). Notably, removal of the extreme carboxyl terminal 26 residues in OspG completely abolished its interaction with ubiquitin (Fig. 3C), suggesting that the carboxyl terminal region following the kinase-like domain in OspG is essential for ubiquitin binding. Mutation of two leucine residues (L190D/L191D) in this region abolishedOspG binding to ubiquitin (Fig. 3C), suggesting the importance of these two residues. Furthermore, when the carboxyl terminal 26, 31, or 35 residues were artificially fused to GST protein and the fusion proteins were assayed for co-precipitation by ubiquitin, none of the three chimeric proteins were capable of binding to ubiquitin (Fig. 3D). These data suggest that the carboxyl terminal helical region in OspG is essential but not sufficient for mediating specific interaction with ubiquitin and the kinase-like domain is also involved in ubiquitin binding.

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