Clostridium difficile toxin A induces expression of the stress-induced early gene product RhoB.
Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly 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 Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts. The expression of RhoB was time-dependent and correlated with the morphological changes of cells. The up-regulation of RhoB was approximately 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. A 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 demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.
Gerhard
Ralf
R
Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. gerhard.ralf@mh-hannover.de
Tatge
Helma
H
Genth
Harald
H
Thum
Thomas
T
Borlak
Jürgen
J
Fritz
Gerhard
G
Just
Ingo
I
D001427:Bacterial Toxins / Q000494:pharmacology*; D018938:Caco-2 Cells; D004768:Enterotoxins / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D011725:Pyridines / Q000494:pharmacology; D013312:Stress, Physiological / Q000235:genetics*; D015854:Up-Regulation / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D007093:Imidazoles; D011725:Pyridines; C041348:tcdA protein, Clostridium difficile; D048051:p38 Mitogen-Activated Protein Kinases; D020743:rhoB GTP-Binding Protein; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole