TcdA^1–1874 lacking the C-terminal repeats still possesses cytotoxic potency.

A) Multidomain structure of C. difficile TcdA and TcdA mutants TcdA^1–1874 and TcdA^1–1101. Full length TcdA consists of the N-terminal glucosyltransferase domain (GTD), the cysteinprotease domain (CPD), the hydrophobic region (HR) acting as transmembrane domain and the C-terminal combined repetitive oligopeptides (CROPs). The CROPs were deleted in TcdA^1–1874. Mutant TcdA^1–1101 exhibits the whole N-terminal domain including the hydrophobic region. B) Cell rounding assay of 3T3 fibroblasts after 90 min of toxin treatment with 1 nM of TcdA, TcdA^1–1874 or TcdA^1–1101, respectively. C) Western blot analysis of toxin-treated cells using antibody either recognizing only non-glucosylated (upper panel) or total Rac1 (lower panel). D) Pre-treatment of 3T3 fibroblasts with 100 nM of Bafilomycin A1, an inhibitor of endosomal acidification, prevents cell rounding of TcdA and TcdA^1–1874 revealing specific cellular uptake of CROP-truncated TcdA. Cells were treated with equipotent concentrations of TcdA (1 nM) and TcdA^1–1874 (10 nM) for 2 h.