Vibrio cholerae strains with mutations in an atypical type I secretion system accumulate RTX toxin intracellularly.
Ontology highlight
ABSTRACT:
SUBMITTER: Boardman BK
PROVIDER: S-EPMC529086 | biostudies-literature | 2004 Dec
REPOSITORIES: biostudies-literature
Publications
Vibrio cholerae strains with mutations in an atypical type I secretion system accumulate RTX toxin intracellularly.
Boardman Bethany Kay BK Satchell Karla J Fullner KJ
Journal of bacteriology 20041201 23
This study shows that the Vibrio cholerae RTX toxin is secreted by a four-component type I secretion system (TISS) encoded by rtxB, rtxD, rtxE, and tolC. ATP-binding site mutations in both RtxB and RtxE blocked secretion, demonstrating that this atypical TISS requires two transport ATPases that may function as a heterodimer. ...[more]
Similar Datasets
Project description: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.
| S-EPMC1868911 | biostudies-literature
Project description: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.
| S-EPMC1932970 | biostudies-literature
Project description:We identify and characterize a gene cluster in El Tor Vibrio cholerae that encodes a cytotoxic activity for HEp-2 cells in vitro. This gene cluster contains four genes and is physically linked to the cholera toxin (CTX) element in the V. cholerae genome. We demonstrate by using insertional mutagenesis that this gene cluster is required for the cytotoxic activity. The toxin, RtxA, resembles members of the RTX (repeats in toxin) toxin family in that it contains a GD-rich repeated motif. Like other RTX toxins, its activity depends on an activator, RtxC, and an associated ABC transporter system, RtxB and RtxD. In V. cholerae strains of the classical biotype, a deletion within the gene cluster removes rtxC and eliminates cytotoxic activity. Other strains, including those of the current cholera pandemic, contain a functional gene cluster and display cytotoxic activity. Thus, the RTX gene cluster in El Tor O1 and O139 strains might have contributed significantly to their emergence. Furthermore, the RTX toxin of V. cholerae may be associated with residual adverse properties displayed by certain live, attenuated cholera vaccines.
| S-EPMC15352 | biostudies-literature
Project description: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 structure/function 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 cross-linking 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.
| S-EPMC470754 | biostudies-literature
Project description:Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX toxin (MARTX(Vc)). Autoprocessing of this toxin by an embedded cysteine protease domain (CPD) is essential for this toxin to induce actin depolymerization in a broad range of cell types. A homologous CPD is also present in the large clostridial toxin TcdB and recent studies showed that inositol hexakisphosphate (Ins(1,2,3,4,5,6)P(6) or InsP(6)) stimulated the autoprocessing of TcdB dependent upon the CPD (Egerer, M., Giesemann, T., Jank, T., Satchell, K. J., and Aktories, K. (2007) J. Biol. Chem. 282, 25314-25321). In this work, the autoprocessing activity of the CPD within MARTX(Vc) is similarly found to be inducible by InsP(6). The CPD is shown to bind InsP(6) (K(d), 0.6 microm), and InsP(6) is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 microm. Processed CPD did not bind InsP(6) indicating that, subsequent to cleavage, the activated CPD may shift to an inactive conformation. To further pursue the mechanism of autoprocessing, conserved residues among 24 identified CPDs were mutagenized. In addition to cysteine and histidine residues that form the catalytic site, 2 lysine residues essential for InsP(6) binding and 5 lysine and arginine residues resulting in loss of activity at low InsP(6) concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP(6) binding pocket and that the binding of InsP(6) stimulates processing by altering the CPD to an activated conformation. After processing, InsP(6) is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP(6).
| S-EPMC3259750 | biostudies-literature
Project description:Cholera is a global disease that has persisted for millennia. The cholera toxin (CT) from Vibrio cholerae is responsible for the clinical symptoms of cholera. This toxin is a hetero-hexamer (AB(5)) complex consisting of a subunit A (CTA) with a pentamer (B(5)) of subunit B (CTB). The importance of the AB(5) complex for pathogenesis is established for the wild type O1 serogroup using known structural and functional data. However, its role is not yet documented in other known serogroups harboring sequence level residue mutations. The sequences for the toxin from different serogroups are available in GenBank (release 177). Sequence analysis reveals mutations at several sequence positions in the toxin across serogroups. Therefore, it is of interest to locate the position of these mutations in the AB(5) structure to infer complex assembly for its functional role in different serogroups. We show that mutations in the CTA are at the solvent exposed regions of the AB(5) complex, whereas those in the CTB are at the CTB/CTB interface of the homo-pentamer complex. Thus, the role of mutations at the CTB/CTB interface for B(5) complex assembly is implied. It is observed that these mutations are often non-synonymous (e.g. polar to non-polar or vice versa). The formation of the AB(5) complex involves inter-subunit residue-residue interactions at the protein-protein interfaces. Hence, these mutations, at the structurally relevant positions, are of importance for the understanding of pathogenesis by several serogroups. This is also of significance in the improvement of recombinant CT protein complex analogs for vaccine design and their use against multiple serogroups.
| S-EPMC3064844 | biostudies-literature
Project description:To study the utility of in vitro-polarized intestinal cell monolayers for modeling Vibrio cholerae-host cell interactions, we added live V. cholerae bacteria to the apical surfaces of polarized T84 cell monolayers and monitored changes in electrical properties. We found that both classical and El Tor strains produce cholera toxin after addition to the monolayer, but induction is most likely due to medium components rather than bacterium-cell interactions. We also found that the RTX toxin is produced by El Tor strains. This toxin caused a loss of the barrier function of the paracellular tight junction that was measured as a decrease in transepithelial resistance. This decrease occurred when bacteria were added to either the apical or basolateral surfaces, indicating that the RTX toxin receptor is expressed on both surfaces. These results are discussed with regard to the applicability of the polarized T84 cell monolayers as an in vitro model of host-pathogen interactions.
| S-EPMC98766 | biostudies-literature
Project description:The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.
| S-EPMC8617504 | biostudies-literature
Project description:The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane. In this study, we report the cloning and sequencing of a DNA fragment from V. cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp. In addition to the two previously described genes, epsE and epsM (M. Sandkvist, V. Morales, and M. Bagdasarian, Gene 123: 81-86, 1993; L. J. Overbye, M. Sandkvist, and M. Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function. Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly. Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli. One of them, a pilin-like protein, EpsG, was analyzed also in V. cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase. We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event. This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V. cholerae and that, when coexpressed in E. coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can.
| S-EPMC179639 | biostudies-other
Project description:The most recently discovered secretion pathway in gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.
| S-EPMC3264300 | biostudies-literature