Clostridium difficile toxin A induces expression of the stress-induced early gene product RhoB

Clostridium difficile toxin A mono-glucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA micro-array revealed the immediate-early gene rhoB as the predominant gene up-regulated in colonic CaCo-2 cells after treatment with toxin A. This toxin A effect was also detectable in epithelial cells such as HT29, and MDCK, as well as NIH 3T3 fibroblasts. Expression of RhoB was time-dependent and correlated with the morphological changes of cells. Up-regulation of RhoB was about 15 fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect. After 8 h a steady state was reached with no further increase in RhoB. The p38 MAPK inhibitor SB202190 reduced the expression of RhoB indicating a participation of the p38 MAPK in this stress response. Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A sparing a pool of potentially active RhoB, as checked by sequential C3bot-catalyzed ADP-ribosylation. Pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells. We desribe for the first time that toxin A not only has the property to inactivate the GTPases RhoA, Rac1 and Cdc42 by glucosylation but also has the property to generate active RhoB that likely contributes to overall picture of intoxication.