FIG 5.

The inhibition of cytopathic effects is due to the inhibition of enzyme activities of TcdB. (A) 7F binds similarly to wild-type TcdB and mutant TcdB-L543A. Ninety-six-well plates were coated with 7F and incubated with serial dilutions of either TcdB or TcdB-L543A (LA). Goat anti-E tag-IgG-HRP was used as a secondary antibody. Overall binding was determined by absorbance at 450 nm (OD₄₅₀). (B) Vero cells were transfected with either 7F, E3, or control (5D) fusion plasmids before exposure to TcdB-L543A (10 ng/ml) and assessed for cytopathic effects over time using a combination of phase-contrast microscopy and fluorescence microscopy. In these pictures, dark cells represent untransfected cells, while red cells represent successfully transfected cells. (C) Quantitative analysis of TcdB-induced cytopathic effects. After TcdB exposure, the percentages of affected cells were determined by counting rounded cells in the total cell population in either untransfected or transfected cells from five random microscopy fields. *, P < 0.05 between transfected and untransfected cells in the same wells.