Escherichia coli serogroup O107/O117 lipopolysaccharide binds and neutralizes Shiga toxin 2

Abstract

The AB(5) toxin Shiga toxin 2 (Stx2) has been implicated as a major virulence factor of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli strains in the progression of intestinal disease to more severe systemic complications. Here, we demonstrate that supernatant from a normal E. coli isolate, FI-29, neutralizes the effect of Stx2, but not the related Stx1, on Vero cells. Biochemical characterization of the neutralizing activity identified the lipopolysaccharide (LPS) of FI-29, a serogroup O107/O117 strain, as the toxin-neutralizing component. LPSs from FI-29 as well as from type strains E. coli O107 and E. coli O117 were able bind Stx2 but not Stx1, indicating that the mechanism of toxin neutralization may involve inhibition of the interaction between Stx2 and the Gb(3) receptor on Vero cells.

FIG. 1.

Effect of E. coli culture supernatants on Stx toxicity to Vero cells. Purified Stx1 or Stx2 was added to culture supernatants to a final concentration of 100 or 250 ng per ml and incubated for 30 min. Vero cells were added and incubated for 3 days. Results are the averages of three trials. An asterisk denotes statistical significance (P < 0.05) compared to the LB medium control.

FIG. 2.

Stx2 coprecipitates with FI-29 LPS. LPS from FI-4 and FI-29 alone, Stx2 from an induced culture of C600::933W, or Stx2 from the induced culture with LPS from FI-4 or FI-29 was centrifuged at 150,000 × g. The presence of Stx2 in the pellets was detected by Western analysis with anti-Stx2 monoclonal antibody 11E10. Stx2 (10 μg) was loaded onto the gel as a positive control for Stx2 detection.

FIG. 3.

Neutralization of Stx2 toxicity to Vero cells by O107 and O117 culture supernatants. Purified Stx2 was added to E. coli O107 or O117 culture supernatants to a final concentration of 100 or 250 ng/ml and incubated for 30 min before incubation with Vero cells for 3 days. Results are the averages of three trials. An asterisk denotes statistical significance (P < 0.05) compared to the LB medium control.

FIG. 4.

Characterization of toxin binding to LPS. A silver stain (A) and toxin affinity blots (B, C, and D) of E. coli LPS are shown. (A) Purified LPS was separated by SDS-PAGE, and the bands were silver stained. (B) LPS bands were transferred to PVDF membranes and probed with Stx2. Bound Stx2 was detected by using anti-Stx2 monoclonal antibody 11E10. Stx2 (10 μg) was loaded and used as a positive control. (C) LPS bands were transferred to PVDF membrane and probed with Stx1. Bound Stx1 was detected by using anti-Stx1 monoclonal antibody 13C4. Stx1 (80 μg) was loaded as a positive control. (D) Concentrated (10×) supernatants of C600, FI-4 (negative control), and FI-29 (positive control) were separated by SDS-PAGE, transferred to PVDF membrane, and probed with Stx2. Bound Stx2 was detected with Stx2 monoclonal antibody 11E10.

FIG. 5.

FI-29 LPS cross-reacts with antibody to serogroups O107 and O117. Purified LPS was separated by SDS-PAGE, and bands were transferred to PVDF membranes. The membranes were probed with anti-O107 or anti-O117 rabbit polyclonal antibody.

FIG. 6.

Antibodies specific to O107 and O117 block Stx2 binding to LPS. Microtiter wells coated with purified LPS (38 nM KDO/well) were incubated with wash buffer (control, no Stx2), Stx2 (15 μg/well), or type O-specific blocking antibody (Ab, as indicated) followed by Stx2. Stx2 binding was determined by using anti-Stx2 monoclonal antibody 11E10 and alkaline phosphatase-conjugated anti-mouse secondary antibody. Results are the average of at least three trials. For each LPS source, an asterisk denotes a statistically higher (P < 0.005) level of Stx2 detected than that for control samples, and a double asterisk denotes a statistically lower (P < 0.05) level of Stx2 detected compared to wells with LPS and Stx2. Between LPS sources, a pound sign denotes a statistically lower (P < 0.02) level of Stx2 binding by E. coli O107 LPS compared to Stx2 binding by FI-29 and O117 LPS types.

FIG. 7.

Structures of the O117 O-polysaccharide (A) and Gb₃ (B).