FIG 2.

E3 and 7F inhibit glucosyltransferase and autoprocessing activities, respectively. (A) E3, but not 7F or other tested V_HHs, affects the glucosyltransferase activity of TcdB. Vero cell lysates were analyzed for Rac1 glucosylation by immunoblotting after exposure to wild-type TcdB and/or E3 or by 7F V_HHs. A monoclonal antibody (clone 102) that binds only to nonglucosylated Rac1 was used as a probe. β-Actin was used as an equal loading control. (B) 7F blocks the autocatalytic cleavage of GTD from full-length TcdB. TcdB was incubated in the presence or absence of 7F and InsP₆. The autocleavage and release of GTD from full-length TcdB were assessed by immunoblotting using a monoclonal antibody against GTD. (C) E3 does not block the autocleavage of GTD from full-length TcdB. (D) The peptide consisting of aa 520 to 543 binds specifically to 7F. Ninety-six-well plates were coated with the peptide consisting of aa 520 to 543 or aa 422 to 439 and incubated with serial dilutions (0.32 to 1,000 ng/ml) of 7F or control V_HH 5D. Goat anti-E tag-IgG-HRP was used as a secondary antibody. (E) The peptide consisting of aa 520 to 543 significantly reduces binding of 7F to GTD. Ninety-six-well plates were coated with 0.5 μg/ml TcdB and incubated with 7F or 5D V_HH alone (100 or 10 ng/ml) or with the V_HHs plus the peptide consisting of aa 520 to 543 or aa 422 to 439. Goat anti-E tag-IgG HRP was used as a secondary antibody. *, P < 0.05; ns, not significant.