Difference in the Cytotoxic Effects of Toxin B fromClostridium difficileStrain VPI 10463 and Toxin B from VariantClostridium difficileStrain 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.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...

Rac1-dependent recruitment of PAK2 to G2phase centrosomes and their roles in the regulation of mitotic entry

“…We employed both the standard Toxin B from the C. difficile strain VPI10463 (TcdB), a broad-spectrum inhibitor of Rho proteins that glucosylates Rho, Rac, and Cdc42 subtype proteins, as well as the variant Toxin B from the C. difficile serotype F strain 1470 (TcdBF). 16,17 TcdBF specifically glucosylates and inactivates Rac1 but not RhoA or Cdc42, as confirmed by a pulldown assay using Rhotekin for RhoA or PAK-CRIB for Rac1 and Cdc42 precipitation (Fig. S5).…”

“…Toxin B from C. difficile strain VPI10463 (TcdB) and the variant Toxin B serotyp F from C. difficile strain 1470 (TcdBF) were purified as described. 16 Anti-Aurora A (#4718), anti-pT288-Aurora A (#3079), anti-PAK1 (#2602), anti-PAK2 (#2608), anti-phospho-T423/402-PAK1/2 (#2601), anti-phospho-S199/S192-PAK1/2 (#2605), anti-phospho-T15-Cdk1 (#9111), anti-PDK1 (#3062), antiCdk1 (#9112), anti-Histone H3 (#9715) were from Cell Signaling Technology. Anti-phospho-S10-histone H3 (3H10, 05-806), antiCyclinB1 (05-373), anti-β-PIX (07-1450), and anti-Rac1 (23A8) were from Millipore.…”

Epithelial cell-cell contacts regulate SRF-mediated transcription via Rac-actin-MAL signalling

“…Tat-C3 is a cell-permeable RhoA-specific ADP-ribosyltransferase (Sahai and Marshall, 2002). TcdB inhibits RhoA and Rac/Cdc42 by glucosylating Thr-37 and Thr-35 within the effector region, respectively (Huelsenbeck et al, 2007). Finally, TcdBF harbours the same receptor-binding and internalisation domain as TcdB, but has an altered glucosyltransferase specificity, targeting selectively Rac and R-Ras (Chaves-Olarte et al, 2003;Huelsenbeck et al, 2007).…”

“…To test directly whether SRF activation upon Ca 2+ withdrawal depends on Rho signalling, we took advantage of the cell-permeable exoenzyme C3 from Clostridium botulinum (Tat-C3), a specific inhibitor of RhoA, and two isoforms of Toxin B from C. difficile, the Rho/Rac/Cdc42-inactivating TcdB and the Rac/R-Rasinactivating TcdBF (Huelsenbeck et al, 2007;Sahai and Marshall, 2002). Ca 2+ was removed from confluent epithelial MDCK cells pretreated with either inhibitor and cells were analysed for SRF activity.…”

Distinct kinetics of (H/K/N)Ras glucosylation and Rac1 glucosylation catalysed by Clostridium sordellii lethal toxin

“…We recently identified the Rac1(Mab 102) antibody as a glucosylation-sensitive antibody allowing the immunoblot detection of Rac1 glucosylation [10,16,17]. This non-radioactive method has been appreciated by many researchers in the field [18,19].…”