Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (ϳ65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.Clostridium difficile produces two large cytotoxins, toxin A (308 kDa) and toxin B (269 kDa). These toxins are the major virulence factors of antibiotic-associated diarrhea and the causative agents of pseudomembranous colitis (1-4). In the cytosol of host cells, both toxins inactivate small GTPases of the Rho family, Rho, Rac, and Cdc42, by monoglucosylation at threonine 37 in Rho and threonine 35 in Rac and Cdc42 (5, 6). Rho GTPases are involved in the organization of the actin cytoskeleton, and treatment of cultured cells with these toxins induces a redistribution of actin filaments, leading to morphological alterations and finally rounding-up of cells (7,8).To reach their substrates in the cytosol, the catalytically active domain of the toxin must cross the cellular membrane. It has been proposed that toxins A and B have a tripartite functional organization (see Fig. 1). The catalytic domain is located at the N terminus of the toxin (9), and the C terminus is believed to bind to the cellular receptor (10 -12). The middle part of the protein harbors a hydrophobic region that is proposed to be involved in the translocation of the toxin across cellular membranes.Ov...

Bordetella dermonecrotic toxin (DNT) causes the deamidation of glutamine 63 of Rho. Here we identified the region of DNT harboring the enzyme activity and compared the toxin with the cytotoxic necrotizing factor 1, which also deamidates Rho. The DNT fragment (⌬DNT) covering amino acid residues 1136 -1451 caused deamidation of RhoA at glutamine 63 as determined by mass spectrometric analysis and by the release of ammonia. In the presence of dansylcadaverine or ethylenediamine, ⌬DNT caused transglutamination of Rho. Deamidase and transglutaminase activities were blocked in the mutant proteins Cys 1292 3 Ala, His 1307 3 Ala, and Lys 1310 3 Ala of ⌬DNT. Deamidation and transglutamination induced by ⌬DNT blocked intrinsic and RhoGTPase-activating protein-stimulated GTPase activity of RhoA. ⌬DNT deamidated and transglutaminated Rac and Cdc42 in the absence and presence of ethylenediamine, respectively. Modification of Rho proteins by ⌬DNT was nucleotide-dependent and did not occur with GTP␥S-loaded GTPases. In contrast to cytotoxic necrotizing factor, which caused the same kinetics of ammonia release in the absence and presence of ethylenediamine, ammonia release by ⌬DNT was largely increased in the presence of ethylenediamine, indicating that ⌬DNT acts primarily as a transglutaminase.Rho GTPases including Rho, Rac, and Cdc42 isoforms are regulators of the actin cytoskeleton and act as molecular switches in a large array of signaling processes (1, 2). The GTPases are the eukaryotic substrates for various bacterial protein toxins (3, 4). C31 -like exoenzymes (e.g. Clostridium botulinum exoenzyme C3) ADP ribosylate RhoA, B, and C at asparagine 41 thereby inhibiting the biological functions of the GTPases (5-7). Large clostridial cytotoxins (e.g. Clostridium difficile toxins A and B) inhibit Rho, Rac, and Cdc42 GTPases by monoglucosylation at threonine 37 and threonine 35, respectively (8, 9). Rho family GTPases are also the targets for the Bordetella dermonecrotic toxin (DNT), which is produced by Bordetella strains (10, 11). DNT induces stress fiber formation, focal adhesion assembly, and tyrosine phosphorylation of focal adhesion kinase and paxillin (10,12,13). Recent studies indicate that DNT causes deamidation of glutamine 63 of RhoA (10). Glutamine 63 is essential for GTP hydrolysis by Rho. Deamidation of glutamine by DNT inhibits the GTPase activity of Rho and renders the Rho protein constitutively active.The same mechanism of Rho activation by deamidation was reported for the cytotoxic necrotizing factor CNF1 from Escherichia coli (14,15). Also CNF deamidates Rho at glutamine 63 and causes similar cytotoxic effects such as multinucleation of cells and stress fiber formation. CNF1 and DNT share a region of homology (amino acid residues 1250 -1351 of DNT) located at the C termini of the toxins (16). Other parts of the protein sequences are not significantly similar. Recently, it was shown that a C-terminal fragment of CNF1 (⌬CNF), covering the region of homology, causes the typical cytotoxic effects after microinje...

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Rho GTPases (e.g. Rho, Rac, and Cdc42) participate in the regulation of the actin cytoskeleton (1, 2). Whereas Rho subtype proteins induce formation of stress fibers and adhesion complexes, Rac is involved in formation of lamellipodia and Cdc42 induces microspikes (3-5). Beside their roles in the organization of the actin cytoskeleton, Rho proteins act as molecular switches in various signal transduction processes (6, 7).Rho proteins are the preferred substrates for several bacterial protein toxins. Exoenzyme C3 from Clostridium botulinum and related C3-like transferases ADP-ribosylate RhoA, B, and C at Asn 41 thereby inhibiting the biological activity of the GTPases (8 -11). Rho proteins are monoglucosylated by members of the family of large clostridial cytotoxins (e.g. Clostridium difficile toxins A and B) (12-14). The toxins modify RhoA at Thr 37 (Thr 35 of Rac and Cdc42), a modification which blocks the interaction of the GTPases with their effectors (15, 16).Rho family GTPases are also the targets for cytotoxic necrotizing factors (CNF) 1 1 and 2 from Escherichia coli and the dermonecrotizing toxin (DNT) produced by various Bordetella species. CNF and DNT are ϳ115 and ϳ165 kDa proteins which share a region of homology at their C termini harboring the enzyme domain of the toxins (17). In culture cells, the toxins induce actin polymerization and inhibit cytokinesis resulting in formation of multinucleated cells (18 -20). Recently it has been reported that CNF and DNT act on Rho GTPases by deamidation of glutamine 63 of RhoA, thereby inhibiting the GTPase activity of Rho. Because Gln 63 is essential for GTP hydrolysis, deamidation causes persistent activation of the GTPase resulting in strong formation of stress fibers of CNF-or DNT-treated cells. In addition to their deamidase activity, both toxins possess in vitro transglutaminase activity to attach primary amines onto Rho GTPases. Substrates of CNF and DNT are Rho subfamily members including Rac and Cdc42.In the present communication the substrate recognition of Rho GTPases by CNF1 and DNT was studied. Using GTPase chimeras of RhoA and Ha-Ras which is not a substrate of the toxins, we identified the switch II region of Rho as being sufficient for recognition by CNF1. Accordingly, a peptide consisting of amino acid residues Asp 59 through Asp 78 of RhoA was deamidated and/or transglutaminated by CNF1. By contrast, the structural requirements for substrate recognition by DNT are more stringent. EXPERIMENTAL PROCEDURESPreparation of Recombinant Proteins-For protein purification, E. coli strains carrying pGEX plasmids with the coding sequence for the respective GTPases, GTPase chimera, CNF1 (either as full-length or as the catalytic C-terminal part (⌬CNF1, amino acid residues 709 -1014), or DNT (the catalytic C-terminal part (⌬DNT, amino acid residues 1136 -1451) were grown in LB medium and induced with 0.2 mM isopropyl-...

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The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is an approximately 97-kDa arginine-specific ADP-ribosyltransferase that is activated by proteolytic cleavage, thereby releasing the active 27-kDa enzyme (MTX(30-264)) and a 70-kDa C-terminal fragment (MTX(265-870)). In solution, the cleaved 70-kDa fragment is still a potent inhibitor of the ADP-ribosyltransferase activity of MTX. Here we studied the interaction of the 70-kDa fragment with the enzyme domain of MTX. Several C-terminal deletions of the 70-kDa fragment inhibited the enzymatic activity of MTX(30-264). However, the IC(50) values were about 2 orders of magnitude higher for the deletions than for the 70-kDa fragment. A peptide covering amino acid residues 265-285 of the holotoxin exhibited the same inhibitory potency as the C-terminal deletions of the 70-kDa fragment. MTX(265-285) contains several acidic residues, of which D273 and D275 were found to be essential for the inhibitory effect. Exchange of these residues in the 70-kDa fragment (MTX(265-870)) reduced its inhibitory potency. Kinetic analysis showed that the peptide MTX(265-285) had no effect on the V(max) of MTX(30-264) but increased the K(m) for NAD. By contrast, the 70-kDa fragment deleted of residues Ile265 through Asn285 inhibited the enzyme activity of MTX(30-264) mainly by decreasing the V(max) of the enzyme. A second binding site for interaction of MTX(265-870) with MTX(30-264) was localized to the C-terminus within the region of residues 750-870. The data support a two-site binding model for inhibition of the ADP-ribosyltransferase activity of MTX(30-264) by the 70-kDa fragment MTX(265-870) with an interaction of amino acid residues 265-285 at the active site and an allosteric inhibition by the C-terminal part of the 70-kDa fragment.