Abstract

Introduction. Clostridium difficile is a pathogen that colonizes the colon in humans when the normal gut microbiome is disrupted. The symptoms of the infection are caused by the toxins TcdA and TcdB. Of these, TcdB is the most relevant in the infectious process, using multiple receptors in mammalian cells. Aim. To evaluate the cytopathic effect of the TcdBNAP1 and TcdBVPI variants in the presence of recombinant RBDs and the differential binding of these to different cell lines. Methodology. We designed two plasmids pET-30a-(+)-TcdB-RBDNAP1/RBDVPI for expression in E. coli and the purification of RBDNAP1 and RBDVPI. The evaluation of these RBDs is performed by fluorescence microscopy in the 3T3, HeLa and CHO cell lines. For the identification of each RBD variant specificity we used the Alexa Fluor 647 dye in a live-cell imaging assay. Otherwise, the cytopathic effect of TcdBNAP1/VPI in the presence of the recombinants RBDs was evaluated through live cell imaging with the Bright Field Filter and considering nuclei staining. Results. We found that the CHO, 3T3 and HeLa cell lines are more susceptible to the VPI variant of toxin B, with the 3T3 line having a higher density of receptors for this variant. 3T3 cell line is also the most resistant to the NAP1 variant. We conclude that the HeLa and the CHO cell lines are good models for toxin detection because their acute response and the 3T3 cell line could be a good model for toxin resistance and attractive for the study of therapeutic targets.