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Infect. Immun. doi:10.1128/IAI.01705-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Difference in the cytotoxic effect between toxin B from Clostridium difficile strain VPI10463 and toxin B from variant Clostridium difficile strain 1470

From the Institut für Toxikologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany, Institute of Pharmacology and Toxicology, University of Ulm Medical Center, D-89081 Ulm, Germany

^* To whom correspondence should be addressed. Email: genth.harald{at}mh-hannover.de.

	Abstract

Glucosylation of Rho(A,B,C), Rac1, and Cdc42 by Clostridium difficile toxin B from strain VPI10463 (TcdB) results in actin re-organization ("cytopathic effect") and apoptosis ("cytotoxic effect"). Toxin B from variant C. difficile strain 1470 serotype (TcdBF) differs from TcdB regarding its substrate proteins, as it glucosylates Rac1 and R-Ras but not Rho(A,B,C) and Cdc42. In this study, we addressed the question if 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, nucleotide fragmentation, and activation of caspase-3; in contrast, TcdBF only induced a marginal cytotoxic effect, suggesting that inactivation of Rho(A,B,C) (but not of Rac1) was required for the cytotoxic effect. The glucosylation of Rac1 correlated to the cytopathic effect of either toxin, suggesting a close connection of both effects. The cytotoxic effect of TcdB was executed by caspase-3, as it was responsive to inhibition by 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 Rho(A,B,C) is required for the cytotoxic effect. In conclusion, the protein substrate specificity of the glucosylating toxins determines their biological activity.

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