Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes

Abstract

Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme caused comparable morphological alteration of CHO cells, which was accompanied by disaggregation of the microfilamental cytoskeleton. The cytotoxic effect of toxin B was correlated with a decrease in C3-catalyzed ADP-ribosylation of the low-molecular-mass GTP-binding protein Rho, which is involved in the regulation of the actin cytoskeleton. We used Xenopus laevis oocytes as a model to study the toxin effect on Rho in more detail. Toxin B treatment of oocytes caused a decrease in subsequent ADP-ribosylation of cytoplasmic Rho by C3. This decrease was observed when toxin B was applied externally or after microinjection. Besides endogenous Rho, microinjected recombinant Rho-glutathione S-transferase fusion protein was affected. Impaired ADP-ribosylation of Rho was neither due to altered guanine nucleotide binding nor to complexation with the guanine nucleotide dissociation inhibitor, which is known to inactivate Rho and to prevent Rho modification by C3. Proteolytical degradation of Rho was excluded by immunoblot analysis. In intact oocytes toxin B caused neither ADP-ribosylation nor phosphorylation of Rho. The data indicate that C. difficile toxin B acts on Rho proteins in Xenopus oocytes to inhibit ADP-ribosylation by C3. It is suggested that toxin B mediates its cytotoxic effect via functional inactivation of Rho.