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Karla J F Satchell - One of the best experts on this subject based on the ideXlab platform.
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structure function analysis of inositol hexakisphosphate induced autoprocessing of the vibrio cholerae multifunctional autoprocessing RTX Toxin
Journal of Biological Chemistry, 2008Co-Authors: Katerina Prochazkova, Karla J F SatchellAbstract:Vibrio cholerae secretes a large virulence-associated multifunctional autoprocessing RTX Toxin (MARTXVc). 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)P6 or InsP6) 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 MARTXVc is similarly found to be inducible by InsP6. The CPD is shown to bind InsP6 (Kd, 0.6 μm), and InsP6 is shown to stimulate intramolecular autoprocessing at both physiological concentrations and as low as 0.01 μm. Processed CPD did not bind InsP6 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 InsP6 binding and 5 lysine and arginine residues resulting in loss of activity at low InsP6 concentrations were identified. Overall, our data support a model in which basic residues located across the CPD structure form an InsP6 binding pocket and that the binding of InsP6 stimulates processing by altering the CPD to an activated conformation. After processing, InsP6 is shown to be recycled, while the cleaved CPD becomes incapable of further binding of InsP6.
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hemolysin and the multifunctional autoprocessing RTX Toxin are virulence factors during intestinal infection of mice with vibrio cholerae el tor o1 strains
Infection and Immunity, 2007Co-Authors: Verena Olivier, Kenneth G Haines, Karla J F SatchellAbstract:The seventh cholera pandemic that started in 1961 was caused by Vibrio cholerae O1 strains of the El Tor biotype. These strains produce the pore-forming Toxin hemolysin, a characteristic used clinically to distinguish classical and El Tor biotypes. Even though extensive in vitro data on the cytolytic activities of hemolysin exist, the connection of hemolysin to virulence in vivo is not well characterized. To study the contribution of hemolysin and other accessory Toxins to pathogenesis, we utilized the model of intestinal infection in adult mice sensitive to the actions of accessory Toxins. In this study, we showed that 4- to 6-week-old streptomycin-fed C57BL/6 mice were susceptible to intestinal infection with El Tor strains, which caused rapid death at high doses. Hemolysin had the predominant role in lethality, with a secondary contribution by the multifunctional autoprocessing RTX (MARTX) Toxin. Cholera Toxin and hemagglutinin/protease did not contribute to lethality in this model. Rapid death was not caused by increased dissemination due to a damaged epithelium since the numbers of CFU recovered from spleens and livers 6 h after infection did not differ between mice inoculated with hemolysin-expressing strains and those infected with non-hemolysin-expressing strains. Although accessory Toxins were linked to virulence, a strain defective in the production of accessory Toxins was still immunogenic since mice immunized with a multiToxin-deficient strain were protected from a subsequent lethal challenge with the wild type. These data suggest that hemolysin and MARTX Toxin contribute to vaccine reactogenicity but that the genes for these Toxins can be deleted from vaccine strains without affecting vaccine efficacy.
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RTX Toxin actin cross linking activity in clinical and environmental isolates of vibrio cholerae
Journal of Clinical Microbiology, 2007Co-Authors: Christina L Cordero, Shanmuga Sozhamannan, Karla J F SatchellAbstract: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.
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autoprocessing of the vibrio cholerae RTX Toxin by the cysteine protease domain
The EMBO Journal, 2007Co-Authors: Kerri Lynn Sheahan, Christina L Cordero, Karla J F SatchellAbstract: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.
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inactivation of small rho gtpases by the multifunctional RTX Toxin from vibrio cholerae
Cellular Microbiology, 2007Co-Authors: Kerri Lynn Sheahan, Karla J F SatchellAbstract:Summary Many 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 cross-linking. 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 Toxin-induced 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.
Joachim Frey - One of the best experts on this subject based on the ideXlab platform.
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Use of recombinant ApxIV in serodiagnosis of Actinobacillus pleuropneumoniae infections and development of an ApxIV ELISA
International Conference on the Epidemiology and Control of Biological Chemical and Physical Hazards in Pigs and Pork, 2014Co-Authors: A. Dreyfus, P. Kuhnert, Joachim FreyAbstract:Actinobacillus pleuropneumoniae (App) is the etiological agent of porcine pleuropneumonia. The virulence of the fifteen serotypes of App is mainly determined by the three major RTX Toxins ApxI, ApxII and ApxIII, which are secreted by the different serotypes in various combinations. A fourth RTX Toxin, ApxIV, is produced by all 15 serotypes specifically during infection of pigs, but not under in vitro conditions. Pigs infected with App show specific antibodies directed against ApxIV. The analysis of sera of experimentally infected pigs revealed that ApxIV-immunoblots detected App infections in the second to third week post infection. We developed an indirect ELISA based on purified recombinant N’-terminal moiety of ApxIV that showed a specificity of 100% and a sensitivity of 93.8%. The prevalidation study of the ApxIV-ELISA revealed that the latter was able to detect App-positive herds, even if clinical and pathological signs of porcine pleuropneumonia were not evident.
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avxa a composite serine protease RTX Toxin of avibacterium paragallinarum
Veterinary Microbiology, 2013Co-Authors: Eliane Kung, Joachim FreyAbstract:Avibacterium paragallinarum, the etiological agent of infectious coryza in chicken, was found to encode a bivalent serine-protease - RTX-porin Toxin named AvxA. This Toxin is encoded on a classical RTX operon structure with the activator gene avxC, the structural serin-protease-RTX Toxin gene avxA, and the genes for a proper type I secretion system avxBD. AvxA is activated by the product of the avxC gene, secreted by the avxBD specified type I secretion system and proteolytically processed leaving a 95 kDa RTX moiety that is found in culture supernatants of A. paragallinarum serovars A, B and C. The RTX moiety of AvxA (AvxA-RTX) is cytotoxic against the avian macrophage like cell line HD11 but not against bovine macrophage cell line BoMac. Purified IgG from hyper-immune rabbit anti-AvxA-RTX serum made by immunization with recombinant AvxA-RTX from a serotype A strain fully neutralizes the cytotoxic activity of recombinant active AvxA-RTX and of A. paragallinarum serotypes A, B and C. This indicates that AvxA is a common major virulence attribute of all A. paragallinarum serotypes.
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distribution of RTX Toxin genes in strains of actinobacillus rossii and pasteurella mairii
Veterinary Microbiology, 2006Co-Authors: Desiree Mayor, Joachim Frey, Bozena M Korczak, Henrik Christensen, Magne Bisgaard, Peter KuhnertAbstract:Strains of [Actinobacillus] rossii, [Pasteurella] mairii and [Pasteurella] aerogenes can be isolated from abortion in swine. The RTX Toxin Pax has previously been found only in those [P.] aerogenes strains isolated from abortion. Nothing is known about RTX Toxins in field isolates of the other two species. To gain insight into the distribution of selected RTX Toxin genes and their association with abortion, PCR screening for the pax, apxII and apxIII operons on 21 [A.] rossii and seven [P.] mairii isolates was done. Since species can be phenotypically misidentified, the study was backed up by a phylogenetic analysis of all strains based on 16S rRNA, rpoB and infB genes. The pax gene was detected in all [P.] mairii but not in [A.] rossii strains. No apx genes were found in [P.] mairii but different gene combinations for apx were detected in [A.] rossii strains. Most of these strains were positive for apxIII, either alone or in combination with apxII. Whereas pax was found to be associated to strains from abortion no such indication could be found with apx in [A.] rossii strains. Phylogenetically [A.] rossii strains formed a heterogeneous cluster separated from Actinobacillus sensu stricto. [P.] mairii strains clustered with [P.] aerogenes but forming a separate branch. The fact that [P.] aerogenes, [P.] mairii and [A.] rossii can phylogenetically clearly be identified and might contain distinct RTX Toxin genes allows their proper diagnosis and will further help to investigate their role as pathogens.
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characterization of the type i secretion system of the RTX Toxin apxii in actinobacillus porcitonsillarum
Veterinary Microbiology, 2005Co-Authors: Peter Kuhnert, Yvonne Schlatter, Joachim FreyAbstract:Strains of Actinobacillus porcitonsillarum are regularly isolated from the tonsils of healthy pigs. A. porcitonsillarum is non pathogenic but phenotypically it strongly resembles the pathogenic species Actinobacillus pleuropneumoniae, thereby interfering with the diagnosis of the latter. A. porcitonsillarum is hemolytic but unlike A. pleuropneumoniae, it contains only apxII genes and not apxI or apxIII genes. In contrast to the truncated apxII operon of A. pleuropneumoniae, which lacks the type I secretion genes BD, characterization of the apxII operon in A. porcitonsillarum revealed that it contains an intact and complete apxII operon. This shows a typical RTX operon structure with the gene arrangement apxIICABD. The region upstream of the apxII operon is also different from that in A. pleuropneumoniae and contains an additional gene, aspC, encoding a putative aspartate aminotransferase. Trans-complementation experiments in Escherichia coli and A. pleuropneumoniae indicated that the entire apxII operon of A. porcitonsillarum is sufficient to express and secrete the ApxIIA Toxin and that the ApxIIA Toxin of A. pleuropneumoniae can be secreted by the type I secretion system encoded by apxIIBD. These findings suggest that the complete apxII operon found in A. porcitonsillarum might be an ancestor of the truncated homologue found in A. pleuropneumoniae. The genetic context of the apxII locus in A. porcitonsillarum and A. pleuropneumoniae suggests that in the latter, the contemporary truncated operon is the result of a recombination event within the species, rather than a horizontal transfer of an incomplete operon.
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phylogenetic relationship of equine actinobacillus species and distribution of RTX Toxin genes among clusters
Veterinary Research, 2003Co-Authors: Peter Kuhnert, Henrik Christensen, Magne Bisgaard, Helene Berthoud, Joachim FreyAbstract:Equine Actinobacillus species were analysed phylogenetically by 16S rRNA gene (rrs) sequencing focusing on the species Actinobacillus equuli, which has recently been subdivided into the non-haemolytic A. equuli subsp. equuli and the haemolytic A. equuli subsp. haemolyticus. In parallel we determined the profile for RTX Toxin genes of the sample of strains by PCR testing for the presence of the A. equuli haemolysin gene aqx, and the Toxin genes apxI, apxII, apxIII and apxIV, which are known in porcine pathogens such as Actinobacillus pleuropneumoniae and Actinobacillus suis. The rrs-based phylogenetic analysis revealed two distinct subclusters containing both A. equuli subsp. equuli and A. equuli subsp. haemolyticus distributed through both subclusters with no correlation to taxonomic classification. Within one of the rrs-based subclusters containing the A. equuli subsp. equuli type strain, clustered as well the porcine Actinobacillus suis strains. This latter is known to be also phenotypically closely related to A. equuli. The Toxin gene analysis revealed that all A. equuli subsp. haemolyticus strains from both rrs subclusters specifically contained the aqx gene while the A. suis strains harboured the genes apxI and apxII. The aqx gene was found to be specific for A. equuli subsp. haemolyticus, since A. equuli subsp. equuli contained no aqx nor any of the other RTX genes tested. The specificity of aqx for the haemolytic equine A. equuli and ApxI and ApxII for the porcine A. suis indicates a role of these RTX Toxins in host species predilection of the two closely related species of bacterial pathogens and allows PCR based diagnostic differentiation of the two.
John J Mekalanos - One of the best experts on this subject based on the ideXlab platform.
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The contribution of accessory Toxins of Vibrio cholerae O1 El Tor to the proinflammatory response in a murine pulmonary cholera model.
Journal of Experimental Medicine, 2002Co-Authors: Karla Jean Fullner, Brian M Meehan, John C. Boucher, Martha A. Hanes, G. Kenneth Haines, Cynthia Walchle, Philippe J. Sansonetti, John J MekalanosAbstract:The contribution of accessory Toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera Toxin genes ( ctxAB ) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-Toxin (RTX) Toxin gene ( RTXA ) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX Toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease ( hapA ) or hemolysin ( hlyA ) did not significantly affect virulence in this model. Compound deletion of ctxAB , hlyA , hapA , and RTXA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-Toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera Toxins.
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vibrio cholerae induced cellular responses of polarized t84 intestinal epithelial cells are dependent on production of cholera Toxin and the RTX Toxin
Infection and Immunity, 2001Co-Authors: Karla Jean Fullner, Wayne I Lencer, John J MekalanosAbstract: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.
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in vivo covalent cross linking of cellular actin by the vibrio cholerae RTX Toxin
The EMBO Journal, 2000Co-Authors: Karla Jean Fullner, John J MekalanosAbstract:Enteric pathogens often export Toxins that elicit diarrhea as a part of the etiology of disease, including Toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated RTXA. Here we investigate the association of RTXA with the cell-rounding effect. We find that V.cholerae exports a large Toxin, RTX (repeats-in-Toxin) Toxin, to culture supernatant fluids and that this Toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX Toxin is not a typical member of the RTX family of pore-forming Toxins. Rather, RTX Toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX Toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the Toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX Toxin, distinguishing it from all other known Toxins.
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In vivo covalent cross‐linking of cellular actin by the Vibrio cholerae RTX Toxin
The EMBO Journal, 2000Co-Authors: Karla Jean Fullner, John J MekalanosAbstract:Enteric pathogens often export Toxins that elicit diarrhea as a part of the etiology of disease, including Toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated RTXA. Here we investigate the association of RTXA with the cell-rounding effect. We find that V.cholerae exports a large Toxin, RTX (repeats-in-Toxin) Toxin, to culture supernatant fluids and that this Toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX Toxin is not a typical member of the RTX family of pore-forming Toxins. Rather, RTX Toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX Toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the Toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX Toxin, distinguishing it from all other known Toxins.
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identification of a vibrio cholerae RTX Toxin gene cluster that is tightly linked to the cholera Toxin prophage
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Karla Jean Fullner, Rebecca A Clayton, Jessica A Sexton, Marc B Rogers, Kerstin E Calia, Stephen B Calderwood, Claire M Fraser, John J MekalanosAbstract: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.
Karla Jean Fullner - One of the best experts on this subject based on the ideXlab platform.
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The contribution of accessory Toxins of Vibrio cholerae O1 El Tor to the proinflammatory response in a murine pulmonary cholera model.
Journal of Experimental Medicine, 2002Co-Authors: Karla Jean Fullner, Brian M Meehan, John C. Boucher, Martha A. Hanes, G. Kenneth Haines, Cynthia Walchle, Philippe J. Sansonetti, John J MekalanosAbstract:The contribution of accessory Toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model. Intranasal administration of an El Tor O1 V. cholerae strain deleted of cholera Toxin genes ( ctxAB ) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage. By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-Toxin (RTX) Toxin gene ( RTXA ) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2. These data suggest that the RTX Toxin contributes to the severity of acute inflammatory responses. Deletions within the genes for either hemagglutinin/protease ( hapA ) or hemolysin ( hlyA ) did not significantly affect virulence in this model. Compound deletion of ctxAB , hlyA , hapA , and RTXA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2. 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant. Thus, the reduced virulence of KFV101 makes it a prototype for multi-Toxin deleted vaccine strains that could be used for protection against V. cholerae without the adverse effects of the accessory cholera Toxins.
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vibrio cholerae induced cellular responses of polarized t84 intestinal epithelial cells are dependent on production of cholera Toxin and the RTX Toxin
Infection and Immunity, 2001Co-Authors: Karla Jean Fullner, Wayne I Lencer, John J MekalanosAbstract: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.
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in vivo covalent cross linking of cellular actin by the vibrio cholerae RTX Toxin
The EMBO Journal, 2000Co-Authors: Karla Jean Fullner, John J MekalanosAbstract:Enteric pathogens often export Toxins that elicit diarrhea as a part of the etiology of disease, including Toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated RTXA. Here we investigate the association of RTXA with the cell-rounding effect. We find that V.cholerae exports a large Toxin, RTX (repeats-in-Toxin) Toxin, to culture supernatant fluids and that this Toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX Toxin is not a typical member of the RTX family of pore-forming Toxins. Rather, RTX Toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX Toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the Toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX Toxin, distinguishing it from all other known Toxins.
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In vivo covalent cross‐linking of cellular actin by the Vibrio cholerae RTX Toxin
The EMBO Journal, 2000Co-Authors: Karla Jean Fullner, John J MekalanosAbstract:Enteric pathogens often export Toxins that elicit diarrhea as a part of the etiology of disease, including Toxins that affect cytoskeletal structure. Recently, we discovered that the intestinal pathogen Vibrio cholerae elicits rounding of epithelial cells that is dependent upon a gene we designated RTXA. Here we investigate the association of RTXA with the cell-rounding effect. We find that V.cholerae exports a large Toxin, RTX (repeats-in-Toxin) Toxin, to culture supernatant fluids and that this Toxin is responsible for cell rounding. Furthermore, we find that cell rounding is not due to necrosis, suggesting that RTX Toxin is not a typical member of the RTX family of pore-forming Toxins. Rather, RTX Toxin causes depolymerization of actin stress fibers and covalent cross-linking of cellular actin into dimers, trimers and higher multimers. This RTX Toxin-specific cross-linking occurs in cells previously rounded with cytochalasin D, indicating that G-actin is the Toxin target. Although several models explain our observations, our simultaneous detection of actin cross-linking and depolymerization points toward a novel mechanism of action for RTX Toxin, distinguishing it from all other known Toxins.
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identification of a vibrio cholerae RTX Toxin gene cluster that is tightly linked to the cholera Toxin prophage
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Karla Jean Fullner, Rebecca A Clayton, Jessica A Sexton, Marc B Rogers, Kerstin E Calia, Stephen B Calderwood, Claire M Fraser, John J MekalanosAbstract: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.
M A Smits - One of the best experts on this subject based on the ideXlab platform.
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genetic map of the actinobacillus pleuropneumoniae RTX Toxin apx operons characterization of the apxiii operons
Infection and Immunity, 1994Co-Authors: R Jansen, J Briaire, E M Kamp, A L J Gielkens, A B M Van Geel, M A SmitsAbstract:Actinobacillus pleuropneumoniae RTX-Toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The Toxin appeared to be a member of the RTX-Toxin family, as are the other two secreted Toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the Toxins in pathogenesis.
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NAD-independent Actinobacillus pleuropneumoniae strains: Production of RTX Toxins and interactions with porcine phagocytes
Veterinary Microbiology, 1994Co-Authors: Freddy Haesebrouck, E M Kamp, M A SmitsAbstract:Actinobacillus pleuropneumoniae RTX Toxin (Apx) production by A. pleuropneumoniae biotype 2 (NAD-independent) serotype 2 strains was studied. Western blot analysis of culture supernatants of all biotype 2 strains tested revealed the presence of a 103 kDa protein which reacted with a monoclonal antibody against ApxIIA. This protein was also recognized by sera of pigs infected with a biotype 2-serotype 2 strain. Furthermore, antibodies that could neutralize ApxIIA were present in these sera. Proteins corresponding to ApxIA or ApxIIIA were not detected. The effects of a biotype 1-serotype 2 and a biotype 2-serotype 2 strain and their metabolites on the oxidative activity of porcine pulmonary alveolar macrophages (PAM) and polymorphonuclear cells (PMN) were compared using a chemiliminescence (CL) technique. Viable bacteria of both biotypes stimulated the production of oxygen radicals by phagocytes. CL responses were higher for the biotype 1 than for the biotype 2 strain. After having reached a peak value, the oxidative activity decreased until a total inhibitions was achieved. Inactivated washed bacteria had no influence on the oxidative activity of phagocytes. In contrast, heat labile factors in culture supernatants of both biotypes stimulated and inhibited the oxidative activity of PAM in a dose-dependent manner. Dilutions of supernatant up to 132 of the biotype 2 strain and up to 1512 of the biotype 1 strain were toxic for PAM, while dilutions from 164 to 1128 of the biotype 2 strain and from 11024to14096 of the biotype 1 strain stimulated the oxidative activity. To evaluate the ability of PAM to eliminate low numbers of the biotype 1-serotype 2 and the biotype 2-serotype 2 strain, 100 CFU of each strain was incubated with 105 PAM. The bacterial growth and the viability of PAM were assessed as a function of time. The biotype 1 strain killed PAM more rapidly and multiplication of the bacteria was higher than for the biotype 2 strain.
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structural analysis of the actinobacillus pleuropneumoniae RTX Toxin i apxi operon
Infection and Immunity, 1993Co-Authors: R Jansen, J Briaire, E M Kamp, A L J Gielkens, M A SmitsAbstract:Actinobacillus pleuropneumoniae-RTX-Toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the Toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural Toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The Toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-Toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed.
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cloning and characterization of the actinobacillus pleuropneumoniae RTX Toxin iii apxiii gene
Infection and Immunity, 1993Co-Authors: R Jansen, J Briaire, E M Kamp, A L J Gielkens, M A SmitsAbstract:To study the role of Actinobacillus pleuropneumoniae-RTX-Toxin III (ApxIII) in the pathogenesis of porcine pleuropneumonia, we cloned and characterized the gene encoding this Toxin. For that purpose, we screened an expression library of genomic DNA of serotype 8 with an ApxIII-specific monoclonal antibody and isolated a 425-bp fragment of an immunoreactive clone. Using this fragment as a probe, we identified and cloned an overlapping chromosomal NsiI restriction fragment of 5.0 kbp. Escherichia coli cells that contained this fragment produced a protein similar to ApxIII. Like ApxIII, the protein had a molecular mass of approximately 120 kDa, was recognized by an ApxIII-specific antibody, killed porcine lung macrophages, and was not lytic for sheep erythrocytes. We concluded from these data that the 5.0-kbp NsiI fragment contained the ApxIII-coding gene. Nucleotide sequence analysis of the 5.0-kbp NsiI fragment revealed the presence of two genes, apxIIIC and apxIIIA. These genes coded for proteins ApxIIIC and ApxIIIA, respectively, which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E. coli. We assumed that the apxIIIA gene coded for the structural RTX Toxin and that the apxIIIC gene coded for its activator. In addition, we found that ApxIII could be secreted from E. coli by the heterologous RTX transporter proteins HlyB and HlyD. The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and 41% identical to that of ApxIIA. We concluded that, beside ApxI and ApxII, ApxIII is the third RTX Toxin produced by A. pleuropneumoniae.
