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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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Related in: MedlinePlus

OspG directly and specifically binds to free ubiquitin.(A) Pulldown of free ubiquitin by OspG. His6-OspG was immobilized onto Ni-NTA beads and the beads were then incubated with GST, GST-ubiquitin, GST-ubiquitin I44A mutant, GST-LC3 or GST-SUMO1. Bound proteins were eluted from the beads and resolved on the SDS-PAGE gel followed by Coomassie blue staining. (B and C) Complex formation of ubiquitin and OspG on the gel filtration column. Purified Flag-His6-ubiquitin and His6-OspG proteins were subjected to Superdex-75 gel filtration chromatography individually or after being mixed together. Shown in (B) are the chromatograms. Aliquots of indicated fractions were resolved on the SDS-PAGE gel stained with Coomassie blue in (C).
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pone-0057558-g002: OspG directly and specifically binds to free ubiquitin.(A) Pulldown of free ubiquitin by OspG. His6-OspG was immobilized onto Ni-NTA beads and the beads were then incubated with GST, GST-ubiquitin, GST-ubiquitin I44A mutant, GST-LC3 or GST-SUMO1. Bound proteins were eluted from the beads and resolved on the SDS-PAGE gel followed by Coomassie blue staining. (B and C) Complex formation of ubiquitin and OspG on the gel filtration column. Purified Flag-His6-ubiquitin and His6-OspG proteins were subjected to Superdex-75 gel filtration chromatography individually or after being mixed together. Shown in (B) are the chromatograms. Aliquots of indicated fractions were resolved on the SDS-PAGE gel stained with Coomassie blue in (C).

Mentions: To confirm the direct interaction between OspG and ubiquitin, purified His6- OspG was used to pull down recombinant GST and GST-ubiquitin. Only GST-ubiquitin was precipitated by His6-OspG and the binding appeared to be highly robust (Fig. 2A). In eukaryotes, there are a large number of ubiquitin-binding modules that confer diverse regulations of ubiquitin signaling. Most of these ubiquitin-binding proteins recognize a critical conserved region on ubiquitin, the I44-centered hydrophobic patch [25], [26]. Interestingly, when I44 of ubiquitin was mutated into Ala, precipitation of this mutant form of ubiquitin by OspG was not observed (Fig. 2A). This result indicates that OspG, similar to most ubiquitin-binding modules in the host, also recognizes the canonical I44 surface of ubiquitin to achieve high-affinity interaction.


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)

OspG directly and specifically binds to free ubiquitin.(A) Pulldown of free ubiquitin by OspG. His6-OspG was immobilized onto Ni-NTA beads and the beads were then incubated with GST, GST-ubiquitin, GST-ubiquitin I44A mutant, GST-LC3 or GST-SUMO1. Bound proteins were eluted from the beads and resolved on the SDS-PAGE gel followed by Coomassie blue staining. (B and C) Complex formation of ubiquitin and OspG on the gel filtration column. Purified Flag-His6-ubiquitin and His6-OspG proteins were subjected to Superdex-75 gel filtration chromatography individually or after being mixed together. Shown in (B) are the chromatograms. Aliquots of indicated fractions were resolved on the SDS-PAGE gel stained with Coomassie blue in (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585378&req=5

pone-0057558-g002: OspG directly and specifically binds to free ubiquitin.(A) Pulldown of free ubiquitin by OspG. His6-OspG was immobilized onto Ni-NTA beads and the beads were then incubated with GST, GST-ubiquitin, GST-ubiquitin I44A mutant, GST-LC3 or GST-SUMO1. Bound proteins were eluted from the beads and resolved on the SDS-PAGE gel followed by Coomassie blue staining. (B and C) Complex formation of ubiquitin and OspG on the gel filtration column. Purified Flag-His6-ubiquitin and His6-OspG proteins were subjected to Superdex-75 gel filtration chromatography individually or after being mixed together. Shown in (B) are the chromatograms. Aliquots of indicated fractions were resolved on the SDS-PAGE gel stained with Coomassie blue in (C).
Mentions: To confirm the direct interaction between OspG and ubiquitin, purified His6- OspG was used to pull down recombinant GST and GST-ubiquitin. Only GST-ubiquitin was precipitated by His6-OspG and the binding appeared to be highly robust (Fig. 2A). In eukaryotes, there are a large number of ubiquitin-binding modules that confer diverse regulations of ubiquitin signaling. Most of these ubiquitin-binding proteins recognize a critical conserved region on ubiquitin, the I44-centered hydrophobic patch [25], [26]. Interestingly, when I44 of ubiquitin was mutated into Ala, precipitation of this mutant form of ubiquitin by OspG was not observed (Fig. 2A). This result indicates that OspG, similar to most ubiquitin-binding modules in the host, also recognizes the canonical I44 surface of ubiquitin to achieve high-affinity interaction.

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