Multiple seropathotypes of verotoxin-producing Escherichia coli (VTEC) disrupt interferon-γ-induced tyrosine phosphorylation of signal transducer and activator of transcription (Stat)-1

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 O157:1-17 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, 6 It, 37 degrees C), and then stimulated with interferon-gamma, (50 ng/mL) for 0.5 h at 37 degrees C. Whole-cell protein extracts of infected cells were collected and prepared for immunoblotting to detect tyrosine phosphorylation of Stat I. The effects of E coli 055 strains, the evolutionary precursors of VTEC, on Statl-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, Statl-tyrosine phosphorylation was significantly reduced after 6h infection of both T84 and HEp-2 cells by VTEC strains of all five seropathotypes. E. coli 055 strains, but not enteropathogenic E. coli (EPEC), also caused inhibition of Statl-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 (0-100nM), but not by treatment with the more specific P13-kinase inhibitor, LY294002. Inhibition of interferon-gamma stimulated Statl-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 Stat I-activation. (c) 2006 Elsevier Ltd. All rights reserved.