Pseudomonas aeruginosa exoenzyme S (ExoS) is a bifunctional cytotoxin. The ADP-ribosyltransferase domain is located within the C terminus part of ExoS. Recent studies showed that the N terminus part of ExoS (amino acid residues 1-234, ExoS(1-234)), which does not possess ADP-ribosyltransferase activity, stimulates cell rounding when transfected or microinjected into eukaryotic cells. Here we studied the effects of ExoS(1-234) on nucleotide binding and hydrolysis by Rho GTPases. ExoS(1-234) (100 -500 nM) did not influence nucleotide exchange of Rho, Rac, and Cdc42 but increased GTP hydrolysis. A similar increase in GTPase activity was stimulated by full-length ExoS. Half-maximal stimulation of GTP hydrolysis by Rho, Rac, and Cdc42 was observed at 10 -11 nM ExoS(1-234), respectively. We identified arginine 146 of ExoS to be essential for the stimulation of GTPase activity of Rho proteins. These data identify ExoS as a GTPase-activating protein for Rho GTPases.Rho GTPases, Rho, Rac, and Cdc42 are involved in the regulation of the actin cytoskeleton by cell membrane-bound receptors and act as molecular switches in a large array of signaling processes (1, 2). Recent studies indicate that the GTPases are the preferred eukaryotic substrates of various bacterial protein toxins and exoenzymes (3,4). C3-like exoenzymes (e.g. Clostridium botulinum exoenzyme C3) ADP-ribosylate RhoA, B, and C at asparagine 41, inhibiting the biological functions of the GTPases (5-7). Large clostridial cytotoxins (e.g. Clostridium difficile toxins A and B) monoglycosylate Rho threonine 37 and Rac and Cdc42 at threonine 35 (8, 9) and induce rounding up of cells and redistribution of the actin cytoskeleton. Escherichia coli cytotoxic necrotizing factors (CNF 1 and 2) 1 and dermonecrotic toxin DNT of Bordetella species (10, 11, 18) activate Rho family GTPases by increasing the lifetime of the active GTP-bound form of the Rho protein. CNF and DNT deamidate and/or transglutaminate glutamine 63 of Rho (glutamine 61 of Rac and Cdc42), 2 which inhibits the intrinsic and GTPase-activating protein (GAP)-stimulated GTP hydrolysis, resulting in a constitutively activated form of the GTPases.Pseudomonas aeruginosa produces two ADP-ribosyltransferases: exotoxin A, which ADP-ribosylates elongation factor 2, or exoenzyme S (ExoS), which ADP-ribosylates Ras (13). ExoS (453 amino acids) is secreted and translocated into eukaryotic target cells by a type III secretion mechanism, which requires bacterial to eukaryotic cell contact. The ADP-ribosyltransferase activity by ExoS is dependent upon a eukaryotic cofactor (FAS, factor activating exoenzyme S), which is a member of the 14-3-3 protein family (14). Ras and several other proteins are preferred eukaryotic substrates of ExoS (15). ExoS ADP-ribosylates Ras at arginine 41 and arginine 128. ADP-ribosylation at arginine 41 blocks the activation of Ras by its guanine nucleotide exchange factor, thereby interfering with Ras-mediated signal transduction (16). The ADP-ribosyltransferase domain is located within the C-...

No abstract

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...

Pseudomonas aeruginosa Exoenzyme S (ExoS) is a bifunctional type-III cytotoxin. The N terminus possesses a Rho GTPase-activating protein (GAP) activity, whereas the C terminus comprises an ADP-ribosyltransferase domain. We investigated whether the ADP-ribosyltransferase activity of ExoS influences its GAP activity. Although the ADP-ribosyltransferase activity of ExoS is dependent upon FAS, a 14-3-3 family protein, factor-activating ExoS (FAS) had no influence on the activity of the GAP domain of ExoS (ExoS-GAP). In the presence of NAD and FAS, the GAP activity of full-length ExoS was reduced about 10-fold, whereas NAD and FAS did not affect the activity of the ExoS-GAP fragment. Using [ 32 P]NAD, ExoS-GAP was identified as a substrate of the ADP-ribosyltransferase activity of ExoS. Site-directed mutagenesis revealed that auto-ADP-ribosylation of Arg-146 of ExoS was crucial for inhibition of GAP activity in vitro.To reveal the auto-ADP-ribosylation of ExoS in intact cells, tetanolysin was used to produce pores in the plasma membrane of Chinese hamster ovary (CHO) cells to allow the intracellular entry of [ 32 P]NAD, the substrate for ADP-ribosylation. After a 3-h infection of CHO cells with Pseudomonas aeruginosa, proteins of 50 and 25 kDa were preferentially ADP-ribosylated. The 50-kDa protein was determined to be auto-ADP-ribosylated ExoS, whereas the 25-kDa protein appeared to represent a group of proteins that included Ras.

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-...