The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking. In this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure͞function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin crosslinking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae.Vibrio vulnificus ͉ VgrG ͉ covalent actin cross-linking

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.

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...

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX Vc ), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G-and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actinbinding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G-and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. The Gram-negative bacterial pathogen Vibrio cholerae is the causative agent of the diarrheal disease cholera. Following ingestion of V. cholerae from contaminated food or water, the bacterium colonizes the host intestine and secretes enterotoxins (1). In addition to cholera toxin, an ADP-ribosylating toxin that stimulates the adenylate cyclase complex, V. cholerae secretes a number of accessory toxins that contribute to pathogenesis, including the multifunctional, autoprocessing RTX toxin of V. cholerae (MARTX Vc ) 4 (2). Active MARTX Vc is a potent toxin produced by the pathogenic O1 El Tor and O139 strains responsible for the current cholera pandemic and a broad range of non-O1, non-O139 clinical and environmental isolates (3). The full-length toxin is Ͼ450,000 Da and comprises a series of glycine-rich repeat regions at the N and C termini and activity domains located within the central portion of the toxin (4). These activity domains carry two distinct cell-rounding activities, one of which leads to cell rounding through inactivation of Rho GTPases (5) and the other by the novel mechanism of covalent actin cross-linking (6).The actin cross-linking domain (ACD) located between the amino acid residues 1963 and 2375 has been identified as the region of MARTX Vc responsible for actin cross-linking activity (7). Recently, a fusion protein of the ACD with the N-terminal portion of Bacillus anthracis lethal factor (LF N ) was tested for actin cross-linking activity both in vivo and in vitro. Purified LF N ACD was translocated to the host cell cytoplasm via the entry mechanism for anthrax toxin, and cross-linked actin proteins were detected in cell lysates. An in vitro actin cross-linking assay was developed to further investigate the role of the ACD in the cross-linking reaction, and it has been demonstrated that LF N ACD directly catalyzes the covalent cross-linking of purified actin in the absence of host protein intermediates. The cross-linking reaction requires both Mg 2ϩ and ATP as cofacto...

Vibrio cholerae strains from diverse O-antigen groups were evaluated for RTX toxin actin cross-linking activity. This study demonstrates that the actin cross-linking domain sequence is present within rtxA in the majority of clinical and environmental isolates tested, and the RTX toxin produced by these strains catalyzes the covalent cross-linking of cellular actin.The causative agent of cholera disease is the gram-negative bacterial pathogen Vibrio cholerae. There are more than 200 O-antigen serogroups of V. cholerae, but only the O1 and O139 strains have been linked to pandemic disease (9). However, non-O1, non-O139 isolates have been associated with a cholera-like disease that leads to clinical symptoms indistinguishable from those of patients infected with O1 and O139 strains (1). Current epidemiological data indicate an increase in the number of pathogenic non-O1, non-O139 isolates, and it has been suggested that the sporadic outbreaks of cholera caused by these strains may be due to bacterial virulence factors other than the well-characterized cholera toxin (CT) and toxin-coregulated pilus (TCP) (1, 6, 15).The V. cholerae RTX (repeats-in-toxin) toxin is encoded by rtxA (12), a gene carried by several non-O1, non-O139 V. cholerae strains linked to cholera disease (3, 5). The activity of the RTX toxin leads to host cell rounding and a novel rearrangement of the actin cytoskeleton-the covalent cross-linking of actin monomers into dimer, trimer, and higher-multimer proteins (8). The region of the toxin responsible for actin cross-linking has been identified as a 412-amino-acid domain present within the 4,545-amino-acid holotoxin (16), and it has been demonstrated that this actin cross-linking domain (ACD) directly catalyzes the cross-linking reaction (4). Actin crosslinking is a fundamental activity of the RTX toxin, and the toxin has been implicated in the pathogenesis of cholera disease (7).Analysis of the rtxA gene in several V. cholerae isolates revealed that the O1 classical strains harbor a deletion in rtxA that eliminates a large portion of the N terminus and inactivates the toxin (3, 12). A previous study by Chow et al. (3) with DNA primers directed against this region indicates that rtxA is distributed across all of the V. cholerae serogroups other than the O1 classical strains. However, a genome sequence comparison of O1 El Tor strain N16961 and O135 strain RC385 showed extensive variation throughout the RTX toxin structure, and among the many differences, the putative RTX toxin in strain RC385 does not carry the ACD. Interestingly, the ACD sequence has been detected in O1 classical isolate O395, despite the fact that this strain contains the N-terminal deletion in rtxA present in all O1 classical strains (The Institute for Genomic Research [www.tigr.org]). The potential for heterogeneity within the toxin structure suggests that an evaluation for the presence of rtxA should include a more thorough investigation of the rtxA sequence, particularly in the region containing the ACD. In addition, it is imp...