The Actin Cross-linking Domain of the Vibrio cholerae RTX Toxin Directly Catalyzes the Covalent Cross-linking of Actin

Abstract

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin crosslinks actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LF N ACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LF N ACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg 2؉ and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.The causative agent of the diarrheal disease cholera is the Gram-negative bacterial pathogen Vibrio cholerae, which is transmitted to the human host following consumption of contaminated food or water. Upon colonization of the upper intestine, V. cholerae produces the major virulence factor cholera toxin, which ADP-ribosylates the ␣-subunit of the G s GTPbinding protein and constitutively activates the adenylate cyclase complex. The subsequent increase in the cAMP levels in intestinal epithelial cells leads to the opening of Cl Ϫ ion channels, resulting in the profuse diarrhea that is the hallmark of cholera. If untreated, the disease can progress to severe dehydration, and case-fatality rates can reach as high as 50% (1).The V. cholerae RTX 4 toxin, which is encoded by rtxA, was discovered through a combination of genomic sequence analysis, genetic mapping, and representational difference analysis (2). The rtxA gene, which is tightly linked to the cholera toxinencoding ctx genes, is deleted in the classical V. cholerae O1 isolates, but is expressed by both the El Tor O1 and O139 strains, which are responsible for the current cholera pandemic (2, 3). The RTX toxin is also produced by non-O1/non-O139 strains, and it has been suggested that the toxin may contribute to the emergence of pathogenic non-O1/non-O139 strains (4).The full-length 4545-amino acid RTX toxin is predicted to be 484,000 Da in size and is secreted from the bacterium by an atypical type I secretion system that requires two transport ATPases (5). Although related to the RTX family of pore-forming toxins, the V. cholerae RTX toxin seems to be the founding member of a new family of RTX exoproteins. These p...

Fatal Diarrheal Disease Caused by <i>Vibrio cholerae</i> O67 in a Patient with Myelodysplastic Syndrome

“…The El Tor type hemolysis toxin exerts a cytolytic effect on intestinal epithelial cells and mucosal cells, consistent with the clinical presentation of Vibrio cholerae non-O1/non-O139 (15). The intestinal toxin, RtxA, is thought to destroy the cytoskeleton and exhibits the same effects as El Tor type hemolysin (16). In this case, the hly and rtxA genes were detected, both of which became presumably potent intestinal virulence factors.…”

MARTX, Multifunctional Autoprocessing Repeats-in-Toxin Toxins

“…Furthermore, cross-linking of actin occurred in cells transiently expressing the domain separated from the remainder of the toxin, a result showing that this domain is sufficient for the actin laddering phenotype (31). Finally, purified ACD has been demonstrated to cross-link actin in vitro in the absence of other cellular proteins, demonstrating that the ACD is an enzyme that directly binds actin and introduces the covalent cross-link (8).…”

The HlyU Protein Is a Positive Regulator of rtxA1 , a Gene Responsible for Cytotoxicity and Virulence in the Human Pathogen Vibrio vulnificus

“…Homologues of rtx genes had also been previously identified on both chromosomes of the V. vulnificus strain YJ016 by in silico analysis showing high identity values (greater than 90%) with the CMCP6 genes (2). The V. cholerae RTX toxin is the second largest single-polypeptide toxin known, and its activity involves the covalent cross-linking of cellular actin, resulting in the depolymerization of actin stress fibers and an increase in paracellular permeability (1,3,28). Whether V. vulnificus RtxA1 behaves in the same way as the V. cholerae homologue has not yet been determined; however, it is clear from our results in this work that V. vulnificus rtxA1 is responsible for the cytotoxicity phenotype of this bacterium, since cytotoxic activity against HeLa cells was greatly decreased in a rtxA1 mutant.…”