Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved in large clostridial glucosylating toxins TcdB, TcdA, TcnA, and TcsL; putative toxins from V. vulnificus, Yersinia sp., Photorhabdus sp., and Xenorhabdus sp.; and a filamentous/hemagglutinin-like protein FhaL from Bordetella sp. In vivo transfection studies and in vitro characterization of purified recombinant protein revealed that this domain from the V. cholerae RTX toxin is an autoprocessing cysteine protease whose activity is stimulated by the intracellular environment. A cysteine point mutation within the RTX holotoxin attenuated actin crosslinking activity suggesting that processing of the toxin is an important step in toxin translocation. Overall, we have uncovered a new mechanism by which large bacterial toxins and proteins deliver catalytic activities to the eukaryotic cell cytosol by autoprocessing after translocation.

SummaryMany bacterial toxins target small Rho GTPases in order to manipulate the actin cytoskeleton. The depolymerization of the actin cytoskeleton by the Vibrio cholerae RTX toxin was previously identified to be due to the unique mechanism of covalent actin crosslinking. However, identification and subsequent deletion of the actin cross-linking domain within the RTX toxin revealed that this toxin has an additional cell rounding activity. In this study, we identified that the multifunctional RTX toxin also disrupts the actin cytoskeleton by causing the inactivation of small Rho GTPases, Rho, Rac and Cdc42. Inactivation of Rho by RTX was reversible in the presence of cycloheximide and by treatment of cells with CNF1 to constitutively activate Rho. These data suggest that RTX targets Rho GTPase regulation rather than affecting Rho GTPase directly. A novel 548-amino-acid region of RTX was identified to be responsible for the toxininduced inactivation of the Rho GTPases. This domain did not carry GAP or phosphatase activities. Overall, these data show that the RTX toxin reversibly inactivates Rho GTPases by a mechanism distinct from other Rho-modifying bacterial toxins.

Extracellular ATP, adenosine (Ado), and adenosine plus homocysteine (Ado/HC) cause apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine and disruption of focal adhesion complexes. Because an increased intracellular ratio of S-adenosylhomocysteine/S-adenosylmethionine favors inhibition of methylation, we hypothesized that Ado/HC might act by inhibition of isoprenylcysteine-O-carboxyl methyltransferase (ICMT). We found that N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) and Nacetyl-S-farnesyl-l-cysteine (AFC), which inhibit ICMT by competing with endogenous substrates for methylation, caused apoptosis. Transient overexpression of ICMT inhibited apoptosis caused by Ado/HC, UV light exposure, or tumor necrosis factor-␣. Because the small GTPase, Ras, is a substrate for ICMT and may modulate apoptosis, we also hypothesized that inhibition of ICMT with Ado/HC or AGGC might cause endothelial apoptosis by altering Ras activation. We found that ICMT inhibition decreased Ras methylation and activity and the activation of the downstream signaling molecules Akt, ERK-1, and ERK-2. Furthermore, overexpression of wild-type or dominant active H-Ras blocked Ado/HC-induced apoptosis. These findings suggest that inhibition of ICMT causes endothelial cell apoptosis by attenuation of Ras GTPase methylation and activation and its downstream antiapoptotic signaling pathway. INTRODUCTIONVascular injury has been implicated in the pathogenesis of disorders such as sepsis and acute respiratory distress syndrome (ARDS). Endothelial cell apoptosis, or programmed cell death, may be important in vascular injury and repair. Apoptotic cells have been identified in increased quantities in the lungs of patients with ARDS, indicating that apoptosis occurs in this syndrome (Polunovsky et al., 1993). Apoptosis can be triggered by disruption of cell-extracellular matrix communication (anoikis) (Frisch and Francis, 1994) or by extracellular factors, such as lipopolysaccharide (Han and Wyche, 1994;Hoyt et al., 1995;Mebmer et al., 1999), tumor necrosis factor (TNF)-␣ (Polunovsky et al., 1994), or UV light (Chatterjee and Wu, 2001). We have previously demonstrated that increased extracellular ATP causes endothelial cell apoptosis after conversion to adenosine and uptake into cells. Moreover, apoptosis caused by intracellular adenosine was enhanced by homocysteine (Dawicki et al., 1997). In subsequent work, we found that increased concentrations of adenosine and homocysteine or inhibition of Sadenosylhomocysteine hydrolase resulted in enhanced levels of S-adenosylhomocysteine (SAH) (Rounds et al., 1998). Because enhanced intracellular concentrations of SAH may result in product inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases (Perna et al., 1997) (Figure 1), we postulated that methyltransferase activity is important in the modulation of endothelial cell apoptosis.Among the methyltransferases is isoprenylcysteine-O-carboxyl methyltransferase (ICMT), the substrates ...

Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or p130(Cas) cDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway.

SummaryActin cross-linking domains (ACDs) are distinct domains found in several bacterial toxins, including the Vibrio cholerae MARTX toxin. The ACD of V. cholerae (ACDVc) catalyses the formation of an irreversible iso-peptide bond between lysine 50 and glutamic acid 270 on two actin molecules in an ATP-and Mg/Mn 2+ -dependent manner. In vivo, cross-linking depletes the cellular pool of G-actin leading to actin cytoskeleton depolymerization. While the actin cross-linking reaction performed by these effector domains has been significantly characterized, the ACDVc catalytic site has remained elusive due to lack of significant homology to known proteins. Using multiple genetic approaches, we have identified regions and amino acids of ACDVc required for full actin cross-linking activity. Then, using these functional data and structural homology predictions, it was determined that several residues demonstrated to be important for ACDVc activity are conserved with active-site residues of the glutamine synthetase family of enzymes. Thus, the ACDs are a family of bacterial toxin effectors that may be evolutionarily related to ligases involved in amino acid biosynthesis.