The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.
The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.
Just
I
I
Institut für Pharmakologie und Toxikologie, Universität des Saarlandes, Federal Republic of Germany.
Wilm
M
M
Selzer
J
J
Rex
G
G
von Eichel-Streiber
C
C
Mann
M
M
Aktories
K
K
D000596:Amino Acids / Q000378:metabolism; D001427:Bacterial Toxins; D016360:Clostridioides difficile / Q000472:pathogenicity*; D004768:Enterotoxins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D020742:rhoA GTP-Binding Protein
D000596:Amino Acids; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D006026:Glycoside Hydrolases; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D005947:Glucose