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Timothy L. Cover - One of the best experts on this subject based on the ideXlab platform.
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Helicobacter pylori Vacuolating Toxin and Gastric Cancer.
Toxins, 2017Co-Authors: Mark S. Mcclain, Amber C. Beckett, Timothy L. CoverAbstract:Helicobacter pylori VacA is a channel-forming Toxin unrelated to other known bacterial Toxins. Most H. pylori strains contain a vacA gene, but there is marked variation among strains in VacA Toxin activity. This variation is attributable to strain-specific variations in VacA amino acid sequences, as well as variations in the levels of VacA transcription and secretion. In this review, we discuss epidemiologic studies showing an association between specific vacA allelic types and gastric cancer, as well as studies that have used animal models to investigate VacA activities relevant to gastric cancer. We also discuss the mechanisms by which VacA-induced cellular alterations may contribute to the pathogenesis of gastric cancer.
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Helicobacter pylori Vacuolating Toxin
Helicobacter pylori Research, 2016Co-Authors: Timothy L. Cover, Robin L. Holland, Steven R BlankeAbstract:The Vacuolating cytoToxin (VacA) was one of the first H. pylori virulence factors identified. All H. pylori strains contain a vacA gene, but there is variation among H. pylori strains in the levels of VacA secretion and activity of VacA proteins. Strains containing allelic types of vacA that produce more active forms of the Toxin are associated with human gastroduodenal disease. Experiments in animal models suggest that VacA may contribute to H. pylori colonization of the stomach, and the Toxin has been linked to gastric epithelial damage. VacA induces a variety of effects in cultured epithelial cells, including alterations in membrane trafficking within the endolysosomal system, mitochondrial dysfunction, and cell death. Most VacA effects on cells are a consequence of intracellular Toxin activities. Unlike most intracellular-acting bacterial Toxins that enzymatically modify target molecules within eukaryotic cells, VacA causes cellular alterations primarily through the formation of intracellular ion-conducting channels. In this chapter, we review the structure and function of VacA, along with the roles of VacA in the pathogenesis of H. pylori-associated diseases.
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Molecular Evolution of the Helicobacter pylori Vacuolating Toxin Gene vacA
Journal of bacteriology, 2010Co-Authors: Kelly A. Gangwer, Timothy L. Cover, D. Borden Lacy, Carrie L. Shaffer, Sebastian Suerbaum, Seth R. BordensteinAbstract:Helicobacter pylori is a genetically diverse organism that is adapted for colonization of the human stomach. All strains contain a gene encoding a secreted, pore-forming Toxin known as VacA. Genetic variation at this locus could be under strong selection as H. pylori adapts to the host immune response, colonizes new human hosts, or inhabits different host environments. Here, we analyze the molecular evolution of VacA. Phylogenetic reconstructions indicate the subdivision of VacA sequences into three main groups with distinct geographic distributions. Divergence of the three groups is principally due to positively selected sequence changes in the p55 domain, a central region required for binding of the Toxin to host cells. Divergent amino acids map to surface-exposed sites in the p55 crystal structure. Comparative phylogenetic analyses of vacA sequences and housekeeping gene sequences indicate that vacA does not share the same evolutionary history as the core genome. Further, rooting the VacA tree with outgroup sequences from the close relative Helicobacter acinonychis reveals that the ancestry of VacA is different from the African origin that typifies the core genome. Finally, sequence analyses of the virulence determinant CagA reveal three main groups strikingly similar to the three groups of VacA sequences. Taken together, these results indicate that positive selection has shaped the phylogenetic structure of VacA and CagA, and each of these virulence determinants has evolved separately from the core genome.
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Analysis of a β-helical region in the p55 domain of Helicobacter pylori Vacuolating Toxin
BMC microbiology, 2010Co-Authors: Susan E. Ivie, Mark S. Mcclain, Holly M. Scott Algood, D. Borden Lacy, Timothy L. CoverAbstract:Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. VacA, a Toxin secreted by H. pylori, is comprised of two domains, designated p33 and p55. Analysis of the crystal structure of the p55 domain indicated that its structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains. Substitution mutations of specific amino acids within the p33 domain abrogate VacA activity, but thus far, it has been difficult to identify small inactivating mutations within the p55 domain. Therefore, we hypothesized that large portions of the p55 domain might be non-essential for Vacuolating Toxin activity. To test this hypothesis, we introduced eight deletion mutations (each corresponding to a single coil within a β-helical segment spanning VacA amino acids 433-628) into the H. pylori chromosomal vacA gene. All eight of the mutant VacA proteins were expressed by the corresponding H. pylori mutant strains and underwent proteolytic processing to yield ~85 kDa passenger domains. Three mutant proteins (VacA Δ484-504, Δ511-536, and Δ517-544) were secreted and induced vacuolation of mammalian cells, which indicated that these β-helical coils were dispensable for Vacuolating Toxin activity. One mutant protein (VacA Δ433-461) exhibited reduced Vacuolating Toxin activity compared to wild-type VacA. Other mutant proteins, including those containing deletions near the carboxy-terminal end of the β-helical region (amino acids Val559-Asn628), exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded. These results indicate that within the β-helical segment of the VacA p55 domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. We propose that non-essential β-helical coils and a carboxy-terminal β-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains.
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Crystal structure of the Helicobacter pylori Vacuolating Toxin p55 domain
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Kelly A. Gangwer, Mark S. Mcclain, Timothy L. Cover, Darren J. Mushrush, Devin L. Stauff, Ben Spiller, D. Borden LacyAbstract:Helicobacter pylori VacA, a pore-forming Toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-Å crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel β-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein Toxins. Notable features of VacA p55 include disruptions in β-sheet contacts that result in five β-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-Å cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation.
Joel B. Baseman - One of the best experts on this subject based on the ideXlab platform.
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Mycoplasma pneumoniae CARDS Toxin Exacerbates Ovalbumin-Induced Asthma-Like Inflammation in BALB/c Mice
2014Co-Authors: Jacqueline J. Coalson, Adriana Chaparro, Molly F. R. Principe, Laura Solis, T. R. Kannan, Joel B. Baseman, Jorge L. Medina, Edward G. Brooks, Vicki T. Winter, Claude Jourdan Le SauxAbstract:Mycoplasma pneumoniae causes a range of airway and extrapulmonary pathologies in humans. Clinically, M. pneumoniae is associated with acute exacerbations of human asthma and a worsening of experimentally induced asthma in mice. Recently, we demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) Toxin, an ADP-ribosylating and Vacuolating Toxin synthesized by M. pneumoniae, is sufficient to induce an asthma-like disease in BALB/cJ mice. To test the potential of CARDS Toxin to exacerbate preexisting asthma, we examined inflammatory responses to recombinant CARDS Toxin in an ovalbumin (OVA) murine model of asthma. Differences in pulmonary inflammatory responses between treatment groups were analyzed by histology, cell differentials and changes in cytokine and chemokine concentrations. Additionally, assessments of airway hyperreactivity were evaluated through direct pulmonary function measurements. Analysis of histology revealed exaggerated cellular inflammation with a strong eosinophilic component in the CARDS Toxin-treated group. Heightened T-helper type-2 inflammatory responses were evidenced by increased expression of IL-4, IL-13, CCL17 and CCL22 corresponding with increased airway hyperreactivity in the CARDS Toxin-treated mice. These data demonstrate that CARDS Toxin can be a causal factor in the worsening of experimental allergic asthma, highlighting the potential importance of CARDS Toxin in the etiology and exacerbation of human asthma.
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mycoplasma pneumoniae community acquired respiratory distress syndrome Toxin expression reveals growth phase and infection dependent regulation
Molecular Microbiology, 2010Co-Authors: Thirumalai R Kannan, Oxana Musatovova, Sowmya Balasubramanian, Marianna Cagle, Jarrat L Jordan, Thomas M Krunkosky, Alan Davis, Robert D Hardy, Joel B. BasemanAbstract:Mycoplasma pneumoniae causes acute and chronic respiratory infections, including tracheobronchitis and community acquired pneumonia, and is linked to asthma and an array of extra-pulmonary disorders. Recently, we identified an ADP-ribosylating and Vacuolating Toxin of M. pneumoniae, designated Community Acquired Respiratory Distress Syndrome (CARDS) Toxin. In this study we analysed CARDS Toxin gene (annotated mpn372) transcription and identified its promoter. We also compared CARDS Toxin mRNA and protein profiles in M. pneumoniae during distinct in vitro growth phases. CARDS Toxin mRNA expression was maximal, but at low levels, during early exponential growth and declined sharply during mid-to-late log growth phases, which was in direct contrast to other mycoplasma genes examined. Between 7% and 10% of CARDS Toxin was localized to the mycoplasma membrane at mid-exponential growth, which was reinforced by immunogold electron microscopy. No CARDS Toxin was released into the medium. Upon M. pneumoniae infection of mammalian cells, increased expression of CARDS Toxin mRNA was observed when compared with SP-4 broth-grown cultures. Further, confocal immunofluorescence microscopy revealed that M. pneumoniae readily expressed CARDS Toxin during infection of differentiated normal human bronchial epithelial cells. Analysis of M. pneumoniae-infected mouse lung tissue revealed high expression of CARDS Toxin per mycoplasma cell when compared with M. pneumoniae cells grown in SP-4 medium alone. Taken together, these studies indicate that CARDS Toxin expression is carefully controlled by environmental cues that influence its transcription and translation. Further, the acceleration of CARDS Toxin synthesis and accumulation in vivo is consistent with its role as a bona fide virulence determinant.
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analysis of pulmonary inflammation and function in the mouse and baboon after exposure to mycoplasma pneumoniae cards Toxin
PLOS ONE, 2009Co-Authors: Jacqueline J. Coalson, Adriana Chaparro, Thirumalai R Kannan, Doug R Hardy, Jay I Peters, Chonnamet Techasaensiri, Angelene M Cantwell, Joel B. BasemanAbstract:Mycoplasma pneumoniae produces an ADP-ribosylating and Vacuolating Toxin known as the CARDS (Community Acquired Respiratory Distress Syndrome) Toxin that has been shown to be cytotoxic to mammalian cells in tissue and organ culture. In this study we tested the ability of recombinant CARDS (rCARDS) Toxin to elicit changes within the pulmonary compartment in both mice and baboons. Animals responded to a respiratory exposure to rCARDS Toxin in a dose and activity-dependent manner by increasing the expression of the pro-inflammatory cytokines IL-1α, 1β, 6, 12, 17, TNF-α and IFN-γ. There was also a dose-dependent increase in several growth factors and chemokines following Toxin exposure including KC, IL-8, RANTES, and G-CSF. Increased expression of IFN-γ was observed only in the baboon; otherwise, mice and baboons responded to CARDS Toxin in a very similar manner. Introduction of rCARDS Toxin to the airways of mice or baboons resulted in a cellular inflammatory response characterized by a dose-dependent early vacuolization and cytotoxicity of the bronchiolar epithelium followed by a robust peribronchial and perivascular lymphocytic infiltration. In mice, rCARDS Toxin caused airway hyper-reactivity two days after Toxin exposure as well as prolonged airway obstruction. The changes in airway function, cytokine expression, and cellular inflammation correlate temporally and are consistent with what has been reported for M. pneumoniae infection. Altogether, these data suggest that the CARDS Toxin interacts extensively with the pulmonary compartment and that the CARDS Toxin is sufficient to cause prolonged inflammatory responses and airway dysfunction.
John L. Telford - One of the best experts on this subject based on the ideXlab platform.
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A Helicobacter pylori Vacuolating Toxin Mutant That Fails To Oligomerize Has a Dominant Negative Phenotype
Infection and immunity, 2006Co-Authors: Marina De Bernard, Silvia Barone, Cesira Galeotti, Roberto Battistutta, Pietro Lupetti, David Mercati, David Skibinski, Christophe Genisset, John L. TelfordAbstract:Most Helicobacter pylori strains secrete a Toxin (VacA) that causes massive vacuolization of target cells and which is a major virulence factor of H. pylori. The VacA amino-terminal region is required for the induction of vacuolization. The aim of the present study was a deeper understanding of the critical role of the N-terminal regions that are protected from proteolysis when VacA interacts with artificial membranes. Using a counterselection system, we constructed an H. pylori strain, SPM 326-Δ49-57, that produces a mutant Toxin with a deletion of eight amino acids in one of these protected regions. VacA Δ49-57 was correctly secreted by H. pylori but failed to oligomerize and did not have any detectable Vacuolating cytotoxic activity. However, the mutant Toxin was internalized normally and stained the perinuclear region of HeLa cells. Moreover, the mutant Toxin exhibited a dominant negative effect, completely inhibiting the Vacuolating activity of wild-type VacA. This loss of activity was correlated with the disappearance of oligomers in electron microscopy. These findings indicate that the deletion in VacA Δ49-57 disrupts the intermolecular interactions required for the oligomerization of the Toxin.
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The Helicobacter pylori Vacuolating Toxin Inhibits T Cell Activation by Two Independent Mechanisms
The Journal of experimental medicine, 2003Co-Authors: John L. Telford, Marianna Boncristiano, Silvia Rossi Paccani, Silvia Barone, Cristina Ulivieri, Laura Patrussi, Dag Ilver, Amedeo Amedei, Mario Milco D'elios, Cosima T. BaldariAbstract:Helicobacter pylori Toxin, VacA, damages the gastric epithelium by erosion and loosening of tight junctions. Here we report that VacA also interferes with T cell activation by two different mechanisms. Formation of anion-specific channels by VacA prevents calcium influx from the extracellular milieu. The transcription factor NF-AT thus fails to translocate to the nucleus and activate key cytokine genes. A second, channel-independent mechanism involves activation of intracellular signaling through the mitogen-activated protein kinases MKK3/6 and p38 and the Rac-specific nucleotide exchange factor, Vav. As a consequence of aberrant Rac activation, disordered actin polymerization is stimulated. The resulting defects in T cell activation may help H. pylori to prevent an effective immune response leading to chronic colonization of its gastric niche.
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Formation of anion-selective channels in the cell plasma membrane by the Toxin VacA of Helicobacter pylori is required for its biological activity
The EMBO journal, 1999Co-Authors: Rino Rappuoli, John L. Telford, Emanuele Papini, Barbara Satin, Cesare Montecucco, Monica Moschioni, Francesco Tombola, Ildikò Szabò, Sandra Brutsche, Mario ZorattiAbstract:The Vacuolating Toxin VacA, a major determinant of Helicobacter pylori-associated gastric diseases, forms anion-selective channels in artificial planar lipid bilayers. Here we show that VacA increases the anion permeability of the HeLa cell plasma membrane and determines membrane depolarization. Electrophysiological and pharmacological approaches indicated that this effect is due to the formation of low-conductance VacA pores in the cell plasma membrane and not to the opening of Ca(2+)- or volume-activated chloride channels. VacA-dependent increase of current conduction both in artificial planar lipid bilayers and in the cellular system was effectively inhibited by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), while2-[(2-cyclopentenyl-6,7dichloro-2, 3-dihydro-2-methyl-1-oxo-1H-inden-5-yl)oxy]acetic acid (IAA-94) was less effective. NPPB inhibited and partially reversed the vacuolation of HeLa cells and the increase of ion conductivity of polarized Madine Darby canine kidney cell monolayers induced by VacA, while IAA-94 had a weaker effect. We conclude that pore formation by VacA accounts for plasma membrane permeabilization and is required for both cell vacuolation and increase of trans-epithelial conductivity.
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Release of Helicobacter pylori Vacuolating cytoToxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released Toxin and vesicles by gastric epithelium
The Journal of pathology, 1999Co-Authors: Roberto Fiocca, Vittorio Ricci, Timothy L. Cover, Vittorio Necchi, Patrizia Sommi, John L. Telford, Enrico SolciaAbstract:The mechanisms by which Helicobacter pylori releases its virulence factors are poorly known. Active secretion has been proposed for some products, including a Vacuolating Toxin (VacA). Outer membrane vesicles represent another mechanism by which some Gram-negative bacteria may release virulence factors. This study sought to localize VacA by immunocytochemistry in H. pylori cells, to determine whether H. pylori produces outer membrane vesicles, and to investigate whether such vesicles might constitute a vehicle for the delivery of bacterial virulence factors to the gastric mucosa. Small (50-300 nm) membrane vesicles were found in H. pylori culture media from both H. pylori strain 60190 and strain CCUG 17874. These vesicles appeared to originate from blebs arising on the bacterial outer membrane. VacA was immunolocalized in the periplasm and outer membrane of intact bacteria and also in outer membrane blebs and vesicles. Both soluble secreted VacA and VacA-containing vesicles bound to, and were internalized by, MKN28 cells and were detectable in the gastric mucosa from H. pylori-infected humans. The release of outer membrane vesicles by H. pylori may represent a mechanism, additional to secretory pathways, for the delivery of bacterial Toxins and antigens to the gastric mucosa.
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Helicobacter pylori Vacuolating Toxin Forms Anion-Selective Channels in Planar Lipid Bilayers: Possible Implications for the Mechanism of Cellular Vacuolation
Biophysical journal, 1999Co-Authors: Francesco Tombola, Rino Rappuoli, John L. Telford, Emanuele Papini, Cesare Montecucco, Marina De Bernard, Ildikò Szabò, Cristina Carlesso, Jean Marc Reyrat, Mario ZorattiAbstract:The Helicobacter pylori VacA Toxin plays a major role in the gastric pathologies associated with this bacterium. When added to cultured cells, VacA induces vacuolation, an effect potentiated by preexposure of the Toxin to low pH. Its mechanism of action is unknown. We report here that VacA forms anion-selective, voltage-dependent pores in artificial membranes. Channel formation was greatly potentiated by acidic conditions or by pretreatment of VacA at low pH. No requirement for particular lipid(s) was identified. Selectivity studies showed that anion selectivity was maintained over the pH range 4.8-12, with the following permeability sequence: Cl- approximately HCO3- > pyruvate > gluconate > K+ approximately Li+ approximately Ba2+ > NH4+. Membrane permeabilization was due to the incorporation of channels with a voltage-dependent conductance in the 10-30 pS range (2 M KCl), displaying a voltage-independent high open probability. Deletion of the NH2 terminus domain (p37) or chemical modification of VacA by diethylpyrocarbonate inhibited both channel activity and vacuolation of HeLa cells without affecting Toxin internalization by the cells. Collectively, these observations strongly suggest that VacA channel formation is needed to induce cellular vacuolation, possibly by inducing an osmotic imbalance of intracellular acidic compartments.
Mark S. Mcclain - One of the best experts on this subject based on the ideXlab platform.
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Helicobacter pylori Vacuolating Toxin and Gastric Cancer.
Toxins, 2017Co-Authors: Mark S. Mcclain, Amber C. Beckett, Timothy L. CoverAbstract:Helicobacter pylori VacA is a channel-forming Toxin unrelated to other known bacterial Toxins. Most H. pylori strains contain a vacA gene, but there is marked variation among strains in VacA Toxin activity. This variation is attributable to strain-specific variations in VacA amino acid sequences, as well as variations in the levels of VacA transcription and secretion. In this review, we discuss epidemiologic studies showing an association between specific vacA allelic types and gastric cancer, as well as studies that have used animal models to investigate VacA activities relevant to gastric cancer. We also discuss the mechanisms by which VacA-induced cellular alterations may contribute to the pathogenesis of gastric cancer.
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Analysis of a β-helical region in the p55 domain of Helicobacter pylori Vacuolating Toxin
BMC microbiology, 2010Co-Authors: Susan E. Ivie, Mark S. Mcclain, Holly M. Scott Algood, D. Borden Lacy, Timothy L. CoverAbstract:Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. VacA, a Toxin secreted by H. pylori, is comprised of two domains, designated p33 and p55. Analysis of the crystal structure of the p55 domain indicated that its structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains. Substitution mutations of specific amino acids within the p33 domain abrogate VacA activity, but thus far, it has been difficult to identify small inactivating mutations within the p55 domain. Therefore, we hypothesized that large portions of the p55 domain might be non-essential for Vacuolating Toxin activity. To test this hypothesis, we introduced eight deletion mutations (each corresponding to a single coil within a β-helical segment spanning VacA amino acids 433-628) into the H. pylori chromosomal vacA gene. All eight of the mutant VacA proteins were expressed by the corresponding H. pylori mutant strains and underwent proteolytic processing to yield ~85 kDa passenger domains. Three mutant proteins (VacA Δ484-504, Δ511-536, and Δ517-544) were secreted and induced vacuolation of mammalian cells, which indicated that these β-helical coils were dispensable for Vacuolating Toxin activity. One mutant protein (VacA Δ433-461) exhibited reduced Vacuolating Toxin activity compared to wild-type VacA. Other mutant proteins, including those containing deletions near the carboxy-terminal end of the β-helical region (amino acids Val559-Asn628), exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded. These results indicate that within the β-helical segment of the VacA p55 domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein secretion or activity, as well as a carboxy-terminal region in which similar alterations result in protein misfolding and impaired secretion. We propose that non-essential β-helical coils and a carboxy-terminal β-helical segment required for proper protein folding and secretion are features shared by numerous autotransporter passenger domains.
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Crystal structure of the Helicobacter pylori Vacuolating Toxin p55 domain
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Kelly A. Gangwer, Mark S. Mcclain, Timothy L. Cover, Darren J. Mushrush, Devin L. Stauff, Ben Spiller, D. Borden LacyAbstract:Helicobacter pylori VacA, a pore-forming Toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-Å crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel β-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein Toxins. Notable features of VacA p55 include disruptions in β-sheet contacts that result in five β-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-Å cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation.
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chapter 24 helicobacter pylori Vacuolating Toxin
The Comprehensive Sourcebook of Bacterial Protein Toxins (Third Edition), 2006Co-Authors: Mark S. Mcclain, Timothy L. CoverAbstract:Helicobacter pylori is a Gram-negative, spiral-shaped bacterium that colonizes the human stomach. Infection by H. pylori is associated with an increased risk for development of peptic ulcer disease, gastric adenocarcinoma, and gastric lymphoma. An exoToxin produced by H. pylori, VacA induces formation of large cytoplasmic vacuoles in cultured epithelial cells. It also contributes to the capacity of H. pylori to colonize the stomach in a murine model of infection. In addition, VacA interferes with the activation and proliferation of T lymphocytes, which might be a factor that enables H. pylori to resist clearance by host immune defenses. This chapter reviews the present understanding of VacA, including its synthesis,genetic diversity, association with disease, activities, interaction with host cells, and structure. Several VacA activities, including vacuole formation and effects on mitochondria, may be attributed to the channel-forming properties of VacA. In this sense, VacA resembles a pore-forming Toxin that acts on intracellular targets. Interestingly, there are numerous VacA proteins for which in vitro activities have not been identified. Further studies of s2-type proteins may lead to the identification of activities shared with si-type Vac A Toxins that are highly relevant in vivo. Alternatively, the s i - and s2-type VacA proteins may have evolved divergent functions suited to the distinct lifestyles of different H. pylori isolates.
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Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori Vacuolating Toxin
Journal of Biological Chemistry, 2003Co-Authors: Mark S. Mcclain, Arlene D. Vinion-dubiel, Hideki Iwamoto, Zhifeng Shao, Yi Li, Gabor Szabo, Timothy L. CoverAbstract:Abstract Helicobacter pylori secretes a Toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxR-VacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. Subsequent experiments examined the capacity of each mutant Toxin to form membrane channels. In a planar lipid bilayer assay, VacA proteins containing G13A, G22A, and G26A mutations formed anion-selective membrane channels, whereas VacA proteins containing P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A, and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. These data indicate that an intact proline residue and an intact G14 XXXG18 motif within the amino-terminal hydrophobic region of VacA are essential for membrane channel formation, and they also provide strong evidence that membrane channel formation is essential for VacA cytotoxicity.
Steven R Blanke - One of the best experts on this subject based on the ideXlab platform.
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Helicobacter pylori Vacuolating Toxin
Helicobacter pylori Research, 2016Co-Authors: Timothy L. Cover, Robin L. Holland, Steven R BlankeAbstract:The Vacuolating cytoToxin (VacA) was one of the first H. pylori virulence factors identified. All H. pylori strains contain a vacA gene, but there is variation among H. pylori strains in the levels of VacA secretion and activity of VacA proteins. Strains containing allelic types of vacA that produce more active forms of the Toxin are associated with human gastroduodenal disease. Experiments in animal models suggest that VacA may contribute to H. pylori colonization of the stomach, and the Toxin has been linked to gastric epithelial damage. VacA induces a variety of effects in cultured epithelial cells, including alterations in membrane trafficking within the endolysosomal system, mitochondrial dysfunction, and cell death. Most VacA effects on cells are a consequence of intracellular Toxin activities. Unlike most intracellular-acting bacterial Toxins that enzymatically modify target molecules within eukaryotic cells, VacA causes cellular alterations primarily through the formation of intracellular ion-conducting channels. In this chapter, we review the structure and function of VacA, along with the roles of VacA in the pathogenesis of H. pylori-associated diseases.
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Remodeling the host environment: modulation of the gastric epithelium by the Helicobacter pylori Vacuolating Toxin (VacA)
Frontiers in cellular and infection microbiology, 2012Co-Authors: Ik Jung Kim, Steven R BlankeAbstract:Virulence mechanisms underlying Helicobacter pylori persistence and disease remain poorly understood, in part, because the factors underlying disease risk are multifactorial and complex. Among the bacterial factors that contribute to the cumulative pathophysiology associated with H. pylori infections, the Vacuolating cytoToxin (VacA) is one of the most important. Analogous to a number of H. pylori genes, the vacA gene exhibits allelic mosaicism, and human epidemiological studies have revealed that several families of Toxin alleles are predictive of more severe disease. Animal model studies suggest that VacA may contribute to pathogenesis in several ways. VacA functions as an intracellular-acting protein exoToxin. However, VacA does not fit the current prototype of AB intracellular-acting bacterial Toxins, which elaborate modulatory effects through the action of an enzymatic domain translocated inside host cells. Rather, VacA may represent an alternative prototype for AB intracellular acting Toxins that modulate cellular homeostasis by forming ion-conducting intracellular membrane channels. Although VacA seems to form channels in several different membranes, one of the most important target sites is the mitochondrial inner membrane. VacA apparently take advantage of an unusual intracellular trafficking pathway to mitochondria, where the Toxin is imported and depolarizes the inner membrane to disrupt mitochondrial dynamics and cellular energy homeostasis as a mechanism for engaging the apoptotic machinery within host cells. VacA remodeling of the gastric environment appears to be fine-tuned through the action of the Type IV effector protein CagA which, in part, limits the cytotoxic effects of VacA in cells colonized by H. pylori.
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Remodeling the Host Environment: Modulation of the Gastric Epithelium by the Helicobacter pylori Vacuolating Toxin (VacA)
Frontiers Media S.A., 2012Co-Authors: Ik-jung Ekim, Steven R BlankeAbstract:Virulence mechanisms underlying Helicobacter pylori persistence and disease remain poorly understood, in part, because disease risk is multifactorial and complex. Among the bacterial factors that contribute to the cumulative pathophysiology associated with H. pylori infections, the Vacuolating cytoToxin (VacA) is one of the most important. Analogous to a number of H. pylori genes, the vacA gene exhibits allelic mosaicism, with several alleles clearly associated with more severe disease. VacA is secreted by Helicobacter pylori as an intracellular-acting exoToxin. However, VacA does not fit the current prototype of AB intracellular-acting bacterial Toxins, which elaborate modulatory effects through the action of an enzymatic domain translocated inside host cells. Rather, VacA may represent an alternative prototype of AB intracellular acting Toxins that modulate membrane-regulated homeostasis by forming ion-conducting intracellular membrane channels. Although VacA seems to form channels in several different membranes, one of the most important target sites is the mitochondrial inner membrane. VacA subverts intracellular trafficking to mitochondria, where the Toxin depolarizes the inner membrane to disrupt mitochondrial dynamics and cellular energy homeostasis as a mechanism for engaging the apoptotic machinery within host cells
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Mutational Analysis of the Helicobacter pylori Vacuolating Toxin Amino Terminus: Identification of Amino Acids Essential for Cellular Vacuolation
Infection and immunity, 2000Co-Authors: Steven R BlankeAbstract:The functional importance of the amino terminus of the Helicobacter pylori Vacuolating cytoToxin (VacA) was investigated by analyzing the relative levels of vacuolation of HeLa cells transfected with plasmids encoding wild-type and mutant forms of the Toxin. Notably, VacA's intracellular activity was found to be sensitive to small truncations and internal deletions at the Toxin's amino terminus. Moreover, alanine-scanning mutagenesis revealed the first VacA point mutations (at proline 9 or glycine 14) that completely abolish the Toxin's intracellular activity.
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A dominant negative mutant of Helicobacter pylori Vacuolating Toxin (VacA) inhibits VacA-induced cell vacuolation
The Journal of biological chemistry, 1999Co-Authors: Arlene D. Vinion-dubiel, Mark S. Mcclain, Hideki Iwamoto, Daniel M. Czajkowsky, Steven R Blanke, Gabor Szabo, Ping Cao, Wayne P. Schraw, Zhifeng ShaoAbstract:Abstract Most Helicobacter pyloristrains secrete a Toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein Toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Δ6–27)) in which this hydrophobic segment was deleted. VacA-(Δ6–27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Δ6–27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Δ6–27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Δ6–27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant Toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Δ6–27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant Toxin exhibited a dominant negative effect, completely inhibiting the Vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant Toxins. We propose a model in which the dominant negative effects of VacA-(Δ6–27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.