No 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.Pathogenic strains of Clostridium difficile cause intestinal infections ranging from mild self-curing diarrhea to the severe form, pseudomembranous colitis (24, 44). These strains produce two toxins, toxin A (TcdA) and toxin B (TcdB) (toxinotype A ϩ B ϩ ). Both toxins are glucosyltransferases that covalently modify Rho, Rac, and Cdc42 (25). These Rho proteins are involved in the control of actin dynamics, cell cycle progression, gene transcription, and vesicle trafficking (10,39,45). The glucosylation site (Thr-37 in RhoA, Thr-35 in Rac1 and Cdc42) is located within the effector region of Rho proteins, resulting in impairment of effector and regulator coupling (16,41,43). Impaired Rho signaling results in reorganization of the actin cytoskeleton (cytopathic effect) and in cell death (cytotoxic effect) (9, 13).Most work on the cytotoxic effect has been performed on TcdA (5, 6, 26, 31). However, the cytotoxic effect of TcdB may also be important for C. difficile-associated disease; e.g., activation of caspase-3 and caspase-9 has been shown to be involved in the cytotoxic effect of TcdB (19,27,29,38).Some C. difficile strains are described as producing solely toxin B and are therefore classified as variant strains (toxinotype A Ϫ B ϩ ) (40). Although toxin B from variant Clostridium difficile strain 1470 serotype F (TcdBF) exhibits an identity of about 93% with TcdB from reference strain VPI 10463 at the amino acid level, TcdBF differs from TcdB with regard to protein substrate specific...

ADP-ribosylation of Rho(A,B,C) by the family of exoenzyme C3-like transferases induces reorganization of the actin cytoskeleton based on inactivation of RhoA. No data are available on the role of RhoB in C3-treated cells. In murine fibroblasts treated with the cell-permeable exoenzyme C3 from Clostridium limosum (C3), an increase in the level of RhoB was observed. This upregulation of RhoB was based on transcriptional activation, as it was responsive to inhibition by actinomycin D and accompanied by activation of the rhoB promoter. Upregulation of RhoB was not observed in cells treated with either the actin ADP-ribosylating C2 toxin from Clostridium botulinum or latrunculin B, suggesting that inactivation of Rho but not actin reorganization was required for the upregulation of RhoB. This notion was confirmed, as the Rho/Rac/Cdc42-glucosylating toxin B from Clostridium difficile (TcdB) but not the Rac/R-Ras-glucosylating variant toxin B from C. difficile strain 1470 serotype F (TcdBF) induced a strong upregulation of RhoB. Upregulation of RhoB was further observed in response to the Rac/(H-,K-,N-,R-)Ras-glucosylating lethal toxin from Clostridium sordellii. The level of active, GTP-bound RhoB was increased in TcdB-treated cells compared to untreated cells (as determined by Rhotekin pull-down assay). In contrast, no active RhoB was found in C3-treated cells. RhoB-GTP was required for the TcdB-induced apoptosis (cytotoxic effect), as this effect was responsive to inhibition by C3. In conclusion, RhoB was upregulated by Rho-/Ras-inactivating toxins, as a consequence of the inactivation of either Rho(A,B,C) or (H-,K-,N-)Ras. In TcdB-treated cells, RhoB escaped its inactivation and was required for the cytotoxic effect.