Clostridium difficile toxin B acts on the GTP-binding protein Rho.
Clostridium difficile toxin B exhibits cytotoxic activity that is characterized by the disruption of the microfilamental cytoskeleton. Here we studied whether the GTP-binding Rho protein, which reportedly participates in the regulation of the actin cytoskeleton, is involved in the toxin action. Toxin B treatment of Chinese hamster ovary cells reveals a time- and concentration-dependent decrease in the ADP-ribosylation of Rho by Clostridium botulinum C3 exoenzyme in the cell lysate. Disruption of the microfilament system induced by C. botulinum C2 toxin or cytochalasin D does not cause impaired ADP-ribosylation of Rho. Toxin B exhibits its effects on Rho not only in intact cells but also when added to cell lysates. Besides endogenous Rho, RhoA-glutathione S-transferase (Rho-GST) fusion protein added to cell lysate showed decreased ADP-ribosylation after toxin B treatment. Immunoblot analysis reveals identical amounts of Rho-GST and no change in molecular mass after toxin B treatment compared with controls. ADP-ribosylation of Rho-GST purified from toxin B-treated cell lysate is inhibited, indicating a modification of Rho itself. Finally, transfection of rhoA DNA under the control of a strong promoter into cells protects them from the activity of toxin B. Altogether, the data indicate that C. difficile toxin B acts directly or indirectly on Rho proteins to inhibit ADP-ribosylation and suggest that the cytotoxic effect of toxin B involves Rho.
Just
I
I
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg-Saar, Germany.
Fritz
G
G
Aktories
K
K
Giry
M
M
Popoff
M R
MR
Boquet
P
P
Hegenbarth
S
S
von Eichel-Streiber
C
C
D016475:3T3 Cells; D036002:ADP Ribose Transferases / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D001905:Botulinum Toxins; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016360:Clostridioides difficile; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000187:drug effects*; D051379:Mice; D008969:Molecular Sequence Data; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein