The Enterotoxin from Clostridium difficile (ToxA) Monoglucosylates the Rho Proteins

Abstract

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.

Application of Mutated Clostridium difficile Toxin A for Determination of Glucosyltransferase-Dependent Effects

“…(GT) activity of TcdA/B, which catalyzes monoglucosylation of the small GTPases Rho, Rac, and Cdc42, is well described (11,12). However, Rho inactivation is not in accordance with the activation of Rho-dependent proinflammatory signal cascades (14).…”

Inhibition of enhanced toxin production by Clostridium difficile in biotin-limited conditions

“…Valine, methionine, tryptophan and glycine were not significantly utilised. When the inhibitory amino acid was added to the medium, asparagine, glutamic acid, glutamine and lysine were consumed in amounts of 1.14, 1.79, 4.75 and 1.53 pmol/ml, respectively. Differences in leucine and iso leucine consumption were observed in media containing inhibitory amino acids.…”

Structural organization of the functional domains of Clostridium difficile toxins A and B

“…The central regions of these toxins have been dubbed "delivery" domains on the basis of the presence of a hydrophobic sequence that could adopt a transmembrane structure during pore formation (12). The N-terminal glucosyltransferase domains are translocated and released into the host cell cytosol, where they can target small GTPases such as RhoA, Rac1, and Cdc42 (13,14). Release is triggered by an autoproteolytic processing event in which eukaryotic inositol hexakisphosphate (InsP6) binds the domain adjacent to the monoglucosyltransferase domain and activates an intramolecular cleavage reaction (15,16).…”