title: Difference in the cytotoxic effects of toxin B from Clostridium difficile strain VPI 10463 and toxin B from variant Clostridium difficile strain 1470.
abstract: Glucosylation of RhoA, Rac1, and Cdc42 by Clostridium difficile toxin B from strain VPI 10463 (TcdB) results in actin reorganization (cytopathic effect) and apoptosis (cytotoxic effect). Toxin B from variant C. difficile strain 1470 serotype F (TcdBF) differs from TcdB with regard to substrate proteins, as it glucosylates Rac1 and R-Ras but not RhoA and Cdc42. In this study, we addressed the question of whether the cellular effects of the toxins depend on their protein substrate specificity. Rat basophilic leukemia (RBL) cells were synchronized using the thymidine double-block technique. We show that cells were most sensitive to the cytotoxic effect of TcdB in S phase, as analyzed in terms of phosphatidyl serine externalization, fragmentation of nuclei, and activation of caspase-3; in contrast, TcdBF induced only a marginal cytotoxic effect, suggesting that inactivation of RhoA (but not of Rac1) was required for the cytotoxic effect. The glucosylation of Rac1 was correlated to the cytopathic effect of either toxin, suggesting a close connection of the two effects. The cytotoxic effect of TcdB was executed by caspase-3, as it was responsive to inhibition by acetyl-Asp-Met-Gln-Asp-aldehyde (Ac-DMQD-CHO), an inhibitor of caspase-3. The viability of TcdB-treated RBL cells was reduced, whereas the viability of TcdBF-treated cells was unchanged, further confirming that inactivation of RhoA is required for the cytotoxic effect. In conclusion, the protein substrate specificity of the glucosylating toxins determines their biological activity.
authors.affiliation: Institut of Toxicology, Medical School, Hannover, D-30625 Hannover, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Huelsenbeck
authors.forename: Stefanie
authors.initials: S
authors.lastname: Dreger
authors.forename: Ralf
authors.initials: R
authors.lastname: Gerhard
authors.forename: Holger
authors.initials: H
authors.lastname: Barth
authors.forename: Ingo
authors.initials: I
authors.lastname: Just
authors.forename: Harald
authors.initials: H
authors.lastname: Genth
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism / Q000633:toxicity*; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D001491:Basophils / Q000166:cytology* / Q000378:metabolism; D053148:Caspase 3 / Q000032:analysis; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D048430:Cell Shape; D002470:Cell Survival; D016360:Clostridioides difficile / Q000145:classification / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D053938:DNA Fragmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006031:Glycosylation; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D009842:Oligopeptides; C110922:acetyl-aspartyl-methionyl-glutaminyl-aspartyl-aldehyde; C031815:toxB protein, Clostridium difficile; D053148:Caspase 3; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein