Figure_1.tif (1.22 MB)
Download file

TcdA1–1874 lacking the C-terminal repeats still possesses cytotoxic potency.

Download (0 kB)
posted on 2013-02-20, 19:46 authored by Alexandra Olling, Sebastian Goy, Florian Hoffmann, Helma Tatge, Ingo Just, Ralf Gerhard

A) Multidomain structure of C. difficile TcdA and TcdA mutants TcdA1–1874 and TcdA1–1101. 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 TcdA1–1874. Mutant TcdA1–1101 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, TcdA1–1874 or TcdA1–1101, 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 TcdA1–1874 revealing specific cellular uptake of CROP-truncated TcdA. Cells were treated with equipotent concentrations of TcdA (1 nM) and TcdA1–1874 (10 nM) for 2 h.