THE ENTEROTOXIN FROM CLOSTRIDIUM-DIFFICILE (TOXA) MONOGLUCOSYLATES THE RHO-PROTEINS

The enterotoxin from Clostridium difficile (ToxA) is one of the causat ive agents of the antibiotic-associated pseudomembranous colitis. In c ultured monolayer cells ToxA exhibits cytotoxic activity to induce dis assembly of the actin cytoskeleton, which is accompanied by morphologi cal changes. ToxA-induced depolymerization of actin filaments is corre lated with a decrease in the ADP-ribosylation of the low molecular mas s GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, R. (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 o f a hexose into Rho. From several hexoses tested UDP-glucose selective ly served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated pep tide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (Rho A, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, a nd Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [C-14]glucose, indicating previous glucos ylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA media tes its cytotoxic effects on cells.