Limits...
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.

Show MeSH

Related in: MedlinePlus

High-affinity binding between OspG and ubiquitin conjugates, poly-ubiquitin chains and free ubiquitin.(A) Pulldown of ubiquitin-conjugated proteins by purified GST-OspG. Glutathione-Sepharose beads coated with GST-OspG or GST alone were incubated with lysates of intact 293T cells or MG132- treated 293T cells. Proteins retained on the beads were eluted with SDS loading buffer and separated onto12% SDS-PAGE gels. Shown on the left are anti-ubiquitin immunoblots and on the right are Coomassie blue staining of GST or GST-OspG proteins present on the beads. (B and C) Pulldown of OpsG by K48- or K63-linked poly-ubiquitin chains. Ni-NTA Sepharose beads coated with His6-ubiquitin chains with indicated linkages were incubated with GST or GST-OspG. Proteins retained on the beads were subjected to SDS-PAGE and anti-GST immunoblotting analysis. (D) Pulldown of free ubiquitin by GST-OspG. GST or GST-OspG proteins were immobilized onto Glutathione Sepharose beads and the beads were then incubated with lysates of 293T cells. The interacting proteins eluted from the beads were resolved by 4–20% gradient SDS-PAGE gel and analyzed by anti-ubiquitin immunoblotting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585378&req=5

pone-0057558-g001: High-affinity binding between OspG and ubiquitin conjugates, poly-ubiquitin chains and free ubiquitin.(A) Pulldown of ubiquitin-conjugated proteins by purified GST-OspG. Glutathione-Sepharose beads coated with GST-OspG or GST alone were incubated with lysates of intact 293T cells or MG132- treated 293T cells. Proteins retained on the beads were eluted with SDS loading buffer and separated onto12% SDS-PAGE gels. Shown on the left are anti-ubiquitin immunoblots and on the right are Coomassie blue staining of GST or GST-OspG proteins present on the beads. (B and C) Pulldown of OpsG by K48- or K63-linked poly-ubiquitin chains. Ni-NTA Sepharose beads coated with His6-ubiquitin chains with indicated linkages were incubated with GST or GST-OspG. Proteins retained on the beads were subjected to SDS-PAGE and anti-GST immunoblotting analysis. (D) Pulldown of free ubiquitin by GST-OspG. GST or GST-OspG proteins were immobilized onto Glutathione Sepharose beads and the beads were then incubated with lysates of 293T cells. The interacting proteins eluted from the beads were resolved by 4–20% gradient SDS-PAGE gel and analyzed by anti-ubiquitin immunoblotting.

Mentions: To search for possible OspG-interacting proteins related to ubiquitination, purified GST-tagged OspG protein (GST-OspG) was immobilized onto glutathione beads and incubated with lysates of intact 293T cells or 293T cells pretreated with the proteasome inhibitor MG132. Proteins retained on the beads were then subjected to reducing SDS-PAGE and anti-ubiquitin (P4D1) Western blotting analysis. Unexpectedly, we found that GST-OspG, but not GST alone, could efficiently pull down a large amount of polyubiquitinated proteins from intact 293T cell lysates, as shown by the heavy smear signals recognized by the anti-ubiquitin antibody (Fig. 1A). When cells were pretreated with MG132 to increase the level of polyubiquitinated proteins, the amounts of polyubiquitinated species precipitated by GST-OspG were also significantly increased and such effects were not observed with pulldown assays performed with GST protein alone (Fig. 1A). These data suggest that OspG can specifically bind to polyubiquitinated proteins in eukaryotic host cells.


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)

High-affinity binding between OspG and ubiquitin conjugates, poly-ubiquitin chains and free ubiquitin.(A) Pulldown of ubiquitin-conjugated proteins by purified GST-OspG. Glutathione-Sepharose beads coated with GST-OspG or GST alone were incubated with lysates of intact 293T cells or MG132- treated 293T cells. Proteins retained on the beads were eluted with SDS loading buffer and separated onto12% SDS-PAGE gels. Shown on the left are anti-ubiquitin immunoblots and on the right are Coomassie blue staining of GST or GST-OspG proteins present on the beads. (B and C) Pulldown of OpsG by K48- or K63-linked poly-ubiquitin chains. Ni-NTA Sepharose beads coated with His6-ubiquitin chains with indicated linkages were incubated with GST or GST-OspG. Proteins retained on the beads were subjected to SDS-PAGE and anti-GST immunoblotting analysis. (D) Pulldown of free ubiquitin by GST-OspG. GST or GST-OspG proteins were immobilized onto Glutathione Sepharose beads and the beads were then incubated with lysates of 293T cells. The interacting proteins eluted from the beads were resolved by 4–20% gradient SDS-PAGE gel and analyzed by anti-ubiquitin immunoblotting.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0057558-g001: High-affinity binding between OspG and ubiquitin conjugates, poly-ubiquitin chains and free ubiquitin.(A) Pulldown of ubiquitin-conjugated proteins by purified GST-OspG. Glutathione-Sepharose beads coated with GST-OspG or GST alone were incubated with lysates of intact 293T cells or MG132- treated 293T cells. Proteins retained on the beads were eluted with SDS loading buffer and separated onto12% SDS-PAGE gels. Shown on the left are anti-ubiquitin immunoblots and on the right are Coomassie blue staining of GST or GST-OspG proteins present on the beads. (B and C) Pulldown of OpsG by K48- or K63-linked poly-ubiquitin chains. Ni-NTA Sepharose beads coated with His6-ubiquitin chains with indicated linkages were incubated with GST or GST-OspG. Proteins retained on the beads were subjected to SDS-PAGE and anti-GST immunoblotting analysis. (D) Pulldown of free ubiquitin by GST-OspG. GST or GST-OspG proteins were immobilized onto Glutathione Sepharose beads and the beads were then incubated with lysates of 293T cells. The interacting proteins eluted from the beads were resolved by 4–20% gradient SDS-PAGE gel and analyzed by anti-ubiquitin immunoblotting.
Mentions: To search for possible OspG-interacting proteins related to ubiquitination, purified GST-tagged OspG protein (GST-OspG) was immobilized onto glutathione beads and incubated with lysates of intact 293T cells or 293T cells pretreated with the proteasome inhibitor MG132. Proteins retained on the beads were then subjected to reducing SDS-PAGE and anti-ubiquitin (P4D1) Western blotting analysis. Unexpectedly, we found that GST-OspG, but not GST alone, could efficiently pull down a large amount of polyubiquitinated proteins from intact 293T cell lysates, as shown by the heavy smear signals recognized by the anti-ubiquitin antibody (Fig. 1A). When cells were pretreated with MG132 to increase the level of polyubiquitinated proteins, the amounts of polyubiquitinated species precipitated by GST-OspG were also significantly increased and such effects were not observed with pulldown assays performed with GST protein alone (Fig. 1A). These data suggest that OspG can specifically bind to polyubiquitinated proteins in eukaryotic host cells.

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