title: Multiple seropathotypes of verotoxin-producing Escherichia coli (VTEC) disrupt interferon-gamma-induced tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1.
abstract: Verotoxin-producing Escherichia coli (VTEC) O157:H7 inhibits interferon-gamma-stimulated tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1 in epithelial cells, independent of Verotoxins and the locus of enterocyte effacement pathogenicity island. Although E. coli O157:H7 is the major cause of disease in humans, non-O157:H7 VTEC also cause human disease. However, the virulence properties of non-O157:H7 VTEC are less well characterized. The aims of this study were to define the ability of VTEC strains of differing seropathotypes (classified as A-E) to inhibit interferon-gamma stimulated Stat1-phosphorylation and to further characterize the bacterial-derived inhibitory factor. Confluent T84 and HEp-2 cells were infected with VTEC strains (MOI 100:1, 6h, 37 degrees C), and then stimulated with interferon-gamma (50 ng/mL) for 0.5h at 37 degrees C. Whole-cell protein extracts of infected cells were collected and prepared for immunoblotting to detect tyrosine phosphorylation of Stat1. The effects of E. coli O55 strains, the evolutionary precursors of VTEC, on Stat1-tyrosine phosphorylation were also determined. The effects of isogenic mutants of O-islands 47 and 122 were tested to determine the role of genes encoded on these putative pathogenicity islands in mediating VTEC inhibition of the interferon-gamma-Stat1 signaling cascade. To evaluate potential mechanism(s) of inhibition, VTEC O157:H7-infected cells were treated with pharmacological inhibitors, including, wortmannin and LY294002. Relative to uninfected cells, Stat1-tyrosine phosphorylation was significantly reduced after 6h infection of both T84 and HEp-2 cells by VTEC strains of all five seropathotypes. E. coli O55 strains, but not enteropathogenic E. coli (EPEC), also caused inhibition of Stat1-tyrosine phosphorylation, suggesting that this effect was acquired early in the evolution of VTEC. Stat1-activation did not recover in epithelial cells infected with isogenic mutants of O-islands 47 and 122, indicating that the inhibitory factor was not contained in these genomic regions. Stat1-phosphorylation remained intact when VTEC-infected cells were treated with wortmannin (0-100 nM), but not by treatment with the more specific PI3-kinase inhibitor, LY294002. Inhibition of interferon-gamma stimulated Stat1-tyrosine phosphorylation by VTEC of multiple seropathotypes indicates the presence of a common inhibitory factor that is independent of bacterial virulence in humans. The results of treatment with wortmannin suggest that the bacterial-derived inhibitory factor employs host cell signal transduction to mediate inhibition of Stat1-activation.
authors.affiliation: Research Institute, Hospital for Sick Children, Ont., Canada.
authors.forename: Narveen
authors.initials: N
authors.lastname: Jandu
authors.forename: Songhai
authors.initials: S
authors.lastname: Shen
authors.forename: Mark E
authors.initials: ME
authors.lastname: Wickham
authors.forename: Rohit
authors.initials: R
authors.lastname: Prajapati
authors.forename: Brett B
authors.initials: BB
authors.lastname: Finlay
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Karmali
authors.forename: Philip M
authors.initials: PM
authors.lastname: Sherman
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000145:classification / Q000378:metabolism* / Q000472:pathogenicity; D004927:Escherichia coli Infections / Q000378:metabolism*; D044404:Genomic Islands / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D022603:Shiga Toxins / Q000378:metabolism; D015398:Signal Transduction; D013045:Species Specificity; D014443:Tyrosine / Q000378:metabolism; D014774:Virulence; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D007371:Interferon-gamma; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D050794:STAT1 Transcription Factor; D022603:Shiga Toxins; D014443:Tyrosine; D000077191:Wortmannin; C494079:STAT1 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one