title: The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.
abstract: Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.
authors.affiliation: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan;
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Su
authors.affiliation: Department of Internal Medicine, Hospital and College of Medicine, National Taiwan University, Taipei 100, Taiwan;
authors.forename: Jyh-Chin
authors.initials: JC
authors.lastname: Yang
authors.affiliation: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan;
authors.forename: Haur
authors.initials: H
authors.lastname: Lee
authors.affiliation: Department of Horticulture, National Taiwan University, Taipei 106, Taiwan;
authors.forename: Fuu
authors.initials: F
authors.lastname: Sheu
authors.affiliation: Department of Agricultural Chemistry, National Taiwan University, Taipei 106, Taiwan; and.
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Hsu
authors.affiliation: Graduate Institute of Physiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
authors.forename: Shuei-Liong
authors.initials: SL
authors.lastname: Lin
authors.affiliation: Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; chowip@ntu.edu.tw.
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Chow
mesh_terms: D000818:Animals; D018835:Chaperonin 10 / Q000235:genetics / Q000276:immunology*; D004220:Disulfides / Q000276:immunology*; D057809:HEK293 Cells; D016480:Helicobacter pylori / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology*; D020935:MAP Kinase Signaling System / Q000235:genetics / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D017434:Protein Structure, Tertiary; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology*
chemical_list: C508606:CXCL8 protein, human; D018835:Chaperonin 10; D004220:Disulfides; D016209:Interleukin-8; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4