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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 stimulates the autophosphorylation and ATP hydrolysis activity of OspG.(A) Kinase assay of OspG in the presence or absence of ubiquitin. Purified OspG protein was incubated with [γ-P32] ATP with or without the addition of ubiquitin into the reaction. Myelin basic protein (MBP) was included as the possible artificial substrate. Incorporation of phosphate into the protein was examined by autoradiography of the reaction mixtures resolved on the SDS-PAGE gel as shown. (B) Effects of mutation in the autophosphorylation site of GST-OspG. The autophosphorylated serine residue in the linker region of GST-OspG was mutated into alanine and the mutant protein (S/A) was subjected to the kinase assay in the presence or absence of ubiquitin as described in (A). (C) Effects of mutations in the putative catalytic residues on ubiquitin stimulation of OspG kinase activity. Wild-type or indicated mutant GST-OspG (K53A and D138A) proteins were used for the autophosphorylation assay in the presence or absence of ubiquitin. Autophosphorylation of OspG is shown by autoradiography in the upper panel. Coomassie blue staining of GST-OspG proteins are shown in the lower panel. (D) Autophosphorylation assay of GST-OspG in the presence of GST-tagged ubiquitin, ubiquitin I44A, LC3 or SUMO1. (E) Autophosphorylation assay of GST-OspG in the presence of free ubiquitin, K48- or K63-linked poly-ubiquitin chains. (F) Autophosphorylation assay of GST-OspG, GST-OspG ΔN23, GST-OspG ΔC26 or GST-OspG L190D/L191D mutant. The assays were all performed in the presence of ubiquitin. (G) ATP hydrolysis assay of OspG and effects of addition of ubiquitin. His6-OspG and ubiquitin were incubated together with or without ATP supplementation. Release of the phosphate following the reaction was detected by measuring the phospho-molybdate complex formation on a spectrometer (absorbance at 600 nm).
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pone-0057558-g005: Ubiquitin binding stimulates the autophosphorylation and ATP hydrolysis activity of OspG.(A) Kinase assay of OspG in the presence or absence of ubiquitin. Purified OspG protein was incubated with [γ-P32] ATP with or without the addition of ubiquitin into the reaction. Myelin basic protein (MBP) was included as the possible artificial substrate. Incorporation of phosphate into the protein was examined by autoradiography of the reaction mixtures resolved on the SDS-PAGE gel as shown. (B) Effects of mutation in the autophosphorylation site of GST-OspG. The autophosphorylated serine residue in the linker region of GST-OspG was mutated into alanine and the mutant protein (S/A) was subjected to the kinase assay in the presence or absence of ubiquitin as described in (A). (C) Effects of mutations in the putative catalytic residues on ubiquitin stimulation of OspG kinase activity. Wild-type or indicated mutant GST-OspG (K53A and D138A) proteins were used for the autophosphorylation assay in the presence or absence of ubiquitin. Autophosphorylation of OspG is shown by autoradiography in the upper panel. Coomassie blue staining of GST-OspG proteins are shown in the lower panel. (D) Autophosphorylation assay of GST-OspG in the presence of GST-tagged ubiquitin, ubiquitin I44A, LC3 or SUMO1. (E) Autophosphorylation assay of GST-OspG in the presence of free ubiquitin, K48- or K63-linked poly-ubiquitin chains. (F) Autophosphorylation assay of GST-OspG, GST-OspG ΔN23, GST-OspG ΔC26 or GST-OspG L190D/L191D mutant. The assays were all performed in the presence of ubiquitin. (G) ATP hydrolysis assay of OspG and effects of addition of ubiquitin. His6-OspG and ubiquitin were incubated together with or without ATP supplementation. Release of the phosphate following the reaction was detected by measuring the phospho-molybdate complex formation on a spectrometer (absorbance at 600 nm).

Mentions: Given that no physiological substrates of OspG have been reported, we performed the kinase assay by using a generic artificial substrate myelin basic protein (MBP) and examined both MBP phosphorylation and OspG autophosphorylation either in the absence or presence of ubiquitin. While no MBP phosphorylation was detected, phosphorylation of GST-OspG by itself was observed (Fig. 5A), which confirms the observation made in the previous study [24]. Given that the autophosphorylation signal is rather weak, we further purified MBP-OspG and His6-OspG and assayed their autophosphorylation activity. Interestingly, no autophosphorylation occurred on MBP-OspG and His6-OspG (data not shown). Through mass spectrometry analysis, the autophosphorylation site of GST-OspG was mapped to a serine residue located in the linker region between the GST and OspG (Fig. S1A). This observation not only explains why no “autophosphorylation” occurred on MBP-OspG and His6-OspG, but also indicates that the observed “autophosphorylation” of GST-OspG differs from the classical kinase autophosphorylation that usually occurs on the activation loop. To confirm the kinase activity of OspG, we then assayed two other commercially available artificial substrates, casein from bovine milk and histone from calf thymus. While no phosphorylation of casein was observed, an evident phosphorylation signal was detected in the crude histone extract (Fig. S1B). Taken together, these data provide definitive experimental evidences for the kinase activity of recombinant OspG protein.


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 stimulates the autophosphorylation and ATP hydrolysis activity of OspG.(A) Kinase assay of OspG in the presence or absence of ubiquitin. Purified OspG protein was incubated with [γ-P32] ATP with or without the addition of ubiquitin into the reaction. Myelin basic protein (MBP) was included as the possible artificial substrate. Incorporation of phosphate into the protein was examined by autoradiography of the reaction mixtures resolved on the SDS-PAGE gel as shown. (B) Effects of mutation in the autophosphorylation site of GST-OspG. The autophosphorylated serine residue in the linker region of GST-OspG was mutated into alanine and the mutant protein (S/A) was subjected to the kinase assay in the presence or absence of ubiquitin as described in (A). (C) Effects of mutations in the putative catalytic residues on ubiquitin stimulation of OspG kinase activity. Wild-type or indicated mutant GST-OspG (K53A and D138A) proteins were used for the autophosphorylation assay in the presence or absence of ubiquitin. Autophosphorylation of OspG is shown by autoradiography in the upper panel. Coomassie blue staining of GST-OspG proteins are shown in the lower panel. (D) Autophosphorylation assay of GST-OspG in the presence of GST-tagged ubiquitin, ubiquitin I44A, LC3 or SUMO1. (E) Autophosphorylation assay of GST-OspG in the presence of free ubiquitin, K48- or K63-linked poly-ubiquitin chains. (F) Autophosphorylation assay of GST-OspG, GST-OspG ΔN23, GST-OspG ΔC26 or GST-OspG L190D/L191D mutant. The assays were all performed in the presence of ubiquitin. (G) ATP hydrolysis assay of OspG and effects of addition of ubiquitin. His6-OspG and ubiquitin were incubated together with or without ATP supplementation. Release of the phosphate following the reaction was detected by measuring the phospho-molybdate complex formation on a spectrometer (absorbance at 600 nm).
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Related In: Results  -  Collection

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pone-0057558-g005: Ubiquitin binding stimulates the autophosphorylation and ATP hydrolysis activity of OspG.(A) Kinase assay of OspG in the presence or absence of ubiquitin. Purified OspG protein was incubated with [γ-P32] ATP with or without the addition of ubiquitin into the reaction. Myelin basic protein (MBP) was included as the possible artificial substrate. Incorporation of phosphate into the protein was examined by autoradiography of the reaction mixtures resolved on the SDS-PAGE gel as shown. (B) Effects of mutation in the autophosphorylation site of GST-OspG. The autophosphorylated serine residue in the linker region of GST-OspG was mutated into alanine and the mutant protein (S/A) was subjected to the kinase assay in the presence or absence of ubiquitin as described in (A). (C) Effects of mutations in the putative catalytic residues on ubiquitin stimulation of OspG kinase activity. Wild-type or indicated mutant GST-OspG (K53A and D138A) proteins were used for the autophosphorylation assay in the presence or absence of ubiquitin. Autophosphorylation of OspG is shown by autoradiography in the upper panel. Coomassie blue staining of GST-OspG proteins are shown in the lower panel. (D) Autophosphorylation assay of GST-OspG in the presence of GST-tagged ubiquitin, ubiquitin I44A, LC3 or SUMO1. (E) Autophosphorylation assay of GST-OspG in the presence of free ubiquitin, K48- or K63-linked poly-ubiquitin chains. (F) Autophosphorylation assay of GST-OspG, GST-OspG ΔN23, GST-OspG ΔC26 or GST-OspG L190D/L191D mutant. The assays were all performed in the presence of ubiquitin. (G) ATP hydrolysis assay of OspG and effects of addition of ubiquitin. His6-OspG and ubiquitin were incubated together with or without ATP supplementation. Release of the phosphate following the reaction was detected by measuring the phospho-molybdate complex formation on a spectrometer (absorbance at 600 nm).
Mentions: Given that no physiological substrates of OspG have been reported, we performed the kinase assay by using a generic artificial substrate myelin basic protein (MBP) and examined both MBP phosphorylation and OspG autophosphorylation either in the absence or presence of ubiquitin. While no MBP phosphorylation was detected, phosphorylation of GST-OspG by itself was observed (Fig. 5A), which confirms the observation made in the previous study [24]. Given that the autophosphorylation signal is rather weak, we further purified MBP-OspG and His6-OspG and assayed their autophosphorylation activity. Interestingly, no autophosphorylation occurred on MBP-OspG and His6-OspG (data not shown). Through mass spectrometry analysis, the autophosphorylation site of GST-OspG was mapped to a serine residue located in the linker region between the GST and OspG (Fig. S1A). This observation not only explains why no “autophosphorylation” occurred on MBP-OspG and His6-OspG, but also indicates that the observed “autophosphorylation” of GST-OspG differs from the classical kinase autophosphorylation that usually occurs on the activation loop. To confirm the kinase activity of OspG, we then assayed two other commercially available artificial substrates, casein from bovine milk and histone from calf thymus. While no phosphorylation of casein was observed, an evident phosphorylation signal was detected in the crude histone extract (Fig. S1B). Taken together, these data provide definitive experimental evidences for the kinase activity of recombinant OspG protein.

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