Multiple seropathotypes of verotoxin-producing Escherichia coli (VTEC) disrupt interferon-gamma-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: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.

Identification of four fimbria-encoding genomic islands that are highly specific for verocytotoxin-producing Escherichia coli serotype O157 strains.

Verocytotoxin-producing Escherichia coli causes zoonotic food- or waterborne infection that may be associated with massive outbreaks and with the serious complication of hemolytic uremic syndrome (HUS). Serotypes O157:H7 and O157:NM are more commonly associated with HUS and outbreaks than other serotypes, such as O26:H11. To determine whether a genetic basis exists for why serotype O157:H7/NM causes HUS and outbreaks more often than other serotypes, such as O26:H11, we conducted suppression subtractive hybridization (SSH) between the genomes of the sequenced O157:H7 strain EDL933 and CL1, a clinical serotype O26:H11 isolate. Genes from four EDL933 fimbria-encoding genomic O islands (OIs) (OI-1, -47, -141, and -154) were identified in the SSH library. OI-47 encodes several additional putative virulence factors, including secreted and signaling proteins, a hemolysin locus, a lipoprotein, an ABC transport system, and a lipid biosynthesis locus. The distribution of the OIs was investigated by PCR and Southern hybridization (when PCR was negative) with 69 VTEC strains belonging to 39 different serotypes corresponding to 5 seropathotypes that differ in their disease and epidemic potential. The four OIs described here were distributed almost exclusively in serotypes O157:H7 and O157:NM, which indicates that they may be associated with the ability of these strains to colonize human and/or animal intestinal tracts and to cause epidemic and serious disease more frequently than other serotypes. The occurrence of the four OIs in enteropathogenic E. coli O55:H7 strains is consistent with their vertical inheritance by VTEC O157:H7/NM from this clonally related ancestor.

Evidence for a hybrid genomic island in verocytotoxin-producing Escherichia coli CL3 (serotype O113:H21) containing segments of EDL933 (serotype O157:H7) O islands 122 and 48.

Genomic O island 122 (OI-122) of the verocytotoxin-producing Escherichia coli (VTEC) strain EDL933 contains four putative virulence genes, Z4321, Z4326, Z4332, and Z4333. However, strain CL3 (serotype O113:H21) contains only Z4321, not the other three genes. To determine whether Z4321 is part of a different genomic island in CL3, a region of 27,293 bp up- and downstream of Z4321 was sequenced and found to contain elements of two different EDL933 genomic islands (OI-48 and OI-122) and a Yersinia pestis-like hemolysin/adhesin gene cluster. The region contained OI-48 genes Z1635, Z1636, and Z1637 at the left terminus and Z1641, Z1642, Z1643, and Z1644 at the right. The middle portion consisted of OI-48 gene Z1640, which was separated into three fragments by genomic segments including the Y. pestis cluster and EDL933 OI-122 genes Z4322, Z4321, and Z4318. In a PCR investigation of 36 VTEC strains of different serotypes, intact Z1640 was present in strains of serotypes O157:H7, O26:H11, O103:H2, O111:NM, and O145:NM, which are associated with hemolytic uremic syndrome and outbreaks. In contrast, fragmented Z1640 was seen in strains of nonepidemic serotypes, such as O91:H21 and O113:H21, and in animal serotypes that have not been associated with human disease, indicating that Z1640 might be a virulence gene.

Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease.

The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five "seropathotypes" (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an "incomplete" OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001).