The sialic acid binding SabA adhesin of Helicobacter pylori is essential for nonopsonic activation of human neutrophils

Infiltration of neutrophils and monocytes into the gastric mucosa is a hallmark of chronic gastritis caused by Helicobacter pylori. Certain H. pylori strains nonopsonized stimulate neutrophils to production of reactive oxygen species causing oxidative damage of the gastric epithelium. Here, the contribution of some H. pylori virulence factors, the blood group antigen-binding adhesin BabA, the sialic acid-binding adhesin SabA, the neutrophil-activating protein HP-NAP, and the vacuolating cytotoxin VacA, to the activation of human neutrophils in terms of adherence, phagocytosis, and oxidative burst was investigated. Neutrophils were challenged with wild type bacteria and isogenic mutants lacking BabA, SabA, HP-NAP, or VacA. Mutant and wild type strains lacking SabA had no neutrophil-activating capacity, demonstrating that binding of H. pylori to sialylated neutrophil receptors plays a pivotal initial role in the adherence and phagocytosis of the bacteria and the induction of the oxidative burst. The link between receptor binding and oxidative burst involves a G-protein-linked signaling pathway and downstream activation of phosphatidylinositol 3-kinase as shown by experiments using signal transduction inhibitors. Collectively our data suggest that the sialic acid-binding SabA adhesin is a prerequisite for the nonopsonic activation of human neutrophils and, thus, is a virulence factor important for the pathogenesis of H. pylori infection.     

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation

Most Helicobacter pylori strains 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-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) 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-(Delta6-27) 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-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) 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-(Delta6-27) 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-(Delta6-27) 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.     

Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity

Helicobacter pylori releases VacA both as free-soluble and as outer membrane vesicle (OMV)-associated toxin. In this study, we investigated the amount of VacA released in each of the two forms and the role of each form in VacA-induced cell vacuolation in vitro. We found that: (1) free-soluble toxin accounted for about 75% of released VacA, while the remaining 25% was OMV-associated; (2) although OMV-associated VacA caused a statistically significant vacuolation, virtually all the vacuolating activity of a H. pylori broth culture filtrate was due to free-soluble VacA. While it is widely accepted that OMVs may represent an important vehicle for delivering virulence factors to the gastric mucosa, our results suggest that OMV-associated VacA could play a pathobiological role different from that of free-soluble toxin. This conclusion fits with mounting evidence that VacA exerts a large pattern of pathobiological effects among which cell vacuolation might not be the main one.     

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.     

Evidence for Transepithelial Dendritic Cells in Human H. pylori Active Gastritis

Background:  Despite extensive experimental investigation stressing the importance of bacterial interaction with dendritic cells (DCs), evidence regarding direct interaction of Helicobacter pylori or its virulence products with DCs in the human gastric mucosa is lacking.
Methods:  Human gastric mucosa biopsies, with or without H. pylori infection and active inflammation, were investigated at light and electron microscopy level with immunocytochemical tests for bacterial products (VacA, urease, outer membrane proteins) and DC markers (DC-SIGN, CD11c, CD83) or with the DC-labeling ZnI₂-OsO₄ technique. Parallel tests with cultured DCs were carried out.
Results:  Cells reproducing ultrastructural and cytochemical patterns of DCs were detected in the lamina propria and epithelium of heavily infected and inflamed (but not of normal) mucosa, where DC luminal endings directly contact H. pylori and take up their virulence products. Cytotoxic changes (mitochondrial swelling, cytoplasmic vacuolation, autophagy) were observed in intraepithelial DCs and reproduced in cultured DCs incubated with H. pylori broth culture filtrates to obtain intracellular accumulation of VacA and urease. Granulocytes were also seen to contact and heavily phagocytose luminal H. pylori , while macrophages remained confined to basal epithelium, though taking up bacteria and bacterial products.
Conclusion:  Human DCs can enter H. pylori -infected gastric epithelium, in association with other innate immunity cells, to take up bacteria and their virulence products. This process is likely to be important for bacterial sensing and pertinent immune response; however, it may also generate DC cytotoxic changes potentially hampering their function.     

Protein-tyrosine phosphatase alpha,RPTP alpha,is a Helicobacter pylori VacA receptor

Helicobacter pylori vacuolating cytotoxin, VacA, induces vacuolation, mitochondrial damage, cytochrome c release, and apoptosis of gastric epithelial cells. To detect gastric proteins that serve as VacA receptors, we used VacA co-immunoprecipitation techniques following biotinylation of the cell surface and identified p250, a receptor-like protein-tyrosine phosphatase beta (RPTP beta) as a VacA-binding protein (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). VacA causes vacuolation of G401 cells, a human kidney tumor cell line, although they do not express RPTP beta. By co-immunoprecipitation with VacA, we identified p140 as a potential receptor in those cells. p140 purified by chromatography on a peanut agglutinin affinity matrix contained internal amino acid sequences of RGEENTDYVNASFIDGYRQK and AEGILDVFQTVK, which are identical to those in RPTP alpha. The peptide mass fingerprinting of p140 by time of flight-MS analysis also supported this identification. Treatment of G401 cells with RPTP alpha-morpholino antisense oligonucleotide before exposure to toxin inhibited vacuolation. These data suggest that RPTP alpha acts as a receptor for VacA in G401 cells. Thus, two receptor tyrosine phosphatases, RPTP alpha and RPTP beta, serve as VacA receptors.     

Cellular vacuolation and mitochondrial cytochrome c release are independent outcomes of Helicobacter pylori vacuolating cytotoxin activity that are each dependent on membrane channel formation

Helicobacter pylori vacuolating toxin (VacA) is a secreted toxin that is reported to produce multiple effects on mammalian cells. In this study, we explored the relationship between VacA-induced cellular vacuolation and VacA-induced cytochrome c release from mitochondria. Within intoxicated cells, vacuolation precedes cytochrome c release and occurs at lower VacA concentrations, indicating that cellular vacuolation is not a downstream consequence of cytochrome c release. Conversely, bafilomycin A1 blocks VacA-induced vacuolation but not VacA-induced cytochrome c release, which indicates that cytochrome c release is not a downstream consequence of cellular vacuolation. Acid activation of purified VacA is required for entry of VacA into cells, and correspondingly, acid activation of the toxin is required for both vacuolation and cytochrome c release, which suggests that VacA must enter cells to produce these two effects. Single amino acid substitutions (P9A and G14A) that ablate vacuolating activity and membrane channel-forming activity render VacA unable to induce cytochrome c release. Channel blockers known to inhibit cellular vacuolation and VacA membrane channel activity also inhibit cytochrome c release. These data indicate that cellular vacuolation and mitochondrial cytochrome c release are two independent outcomes of VacA intoxication and that both effects are dependent on the formation of anion-selective membrane channels.     

Helicobacter felis does not stimulate human neutrophil oxidative burst in contrast to 'Gastrospirillum hominis' and Helicobacter pylori

Helicobacter pylori is a human pathogen, whereas the natural hosts for 'Gastrospirillum hominis' and Helicobacter felis are animals. 'G. hominis' is occasionally found to cause infection in humans, whereas H. felis only rarely infects humans. The pathogenesis of H. pylori infection is not completely understood and in order to reveal differences in immune response to the three Helicobacter species, the upregulation of adherence molecule CD11b/CD18, chemotactic activity and oxidative burst response of neutrophils after stimulation with H. pylori, 'G. hominis' and H. felis sonicates, were compared. Like H. pylori, 'G. hominis' and H. felis induced upregulation of CD11b/CD18 and chemotaxis of neutrophils. 'G. hominis' demonstrated a more pronounced upregulation of CD11b/CD18, whereas H. felis was the strongest stimulant of neutrophil chemotaxis. H. felis was unable to stimulate neutrophils to oxidative burst response, whereas 'G. hominis' activated neutrophils in a dose-dependent way similar to H. pylori. 'G. hominis' and H. felis were both able to prime neutrophils for oxidative burst response similar to H. pylori. In conclusion, we observed clear differences in neutrophil responses to different Helicobacter species, which indicates that bacterial virulence factors may be important for the diversity in the pathogenetic outcome of Helicobacter infections.     

Integrin subunit CD18 Is the T-lymphocyte receptor for the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori infection is associated with gastritis, ulcerations, and gastric adenocarcinoma. H. pylori secretes the vacuolating cytotoxin (VacA), a major pathogenicity factor. VacA has immunosuppressive effects, inhibiting interleukin-2 (IL-2) secretion by interference with the T cell receptor/IL-2 signaling pathway at the level of calcineurin, the Ca2+-calmodulin-dependent phosphatase. Here, we show that VacA efficiently enters activated, migrating primary human T lymphocytes by binding to the beta2 (CD18) integrin receptor subunit and exploiting the recycling of lymphocyte function-associated antigen (LFA)-1. LFA-1-deficient Jurkat T cells were resistant to vacuolation and IL-2 modulation, and genetic complementation restored sensitivity to VacA. VacA targeted human, but not murine, CD18 for cell entry, consistent with the species-specific adaptation of H. pylori. Furthermore, expression of human integrin receptors (LFA-1 or Mac-1) in murine T cells resulted in VacA-mediated cellular vacuolation. Thus, H. pylori co-opts CD18 as a VacA receptor on human T lymphocytes to subvert the host immune response.     

Association of Helicobacter pylori vacuolating toxin (VacA) with lipid rafts

A variety of extracellular ligands and pathogens interact with raft domains in the plasma membrane of eukaryotic cells. In this study, we examined the role of lipid rafts and raft-associated glycosylphosphatidylinositol (GPI)-anchored proteins in the process by which Helicobacter pylori vacuolating toxin (VacA) intoxicates cells. We first investigated whether GPI-anchored proteins are required for VacA toxicity by analyzing wild-type Chinese hamster ovary (CHO) cells and CHO-LA1 mutant cells that are defective in production of GPI-anchored proteins. Whereas wild-type and mutant cells differed markedly in susceptibility to aerolysin (a bacterial toxin that binds to GPI-anchored proteins), they were equally susceptible to VacA. We next determined whether VacA physically associates with lipid rafts. CHO or HeLa cells were incubated with VacA, and Triton-insoluble membranes then were separated by sucrose density gradient centrifugation. Immunoblot analysis revealed that a substantial proportion of cell-associated toxin was associated with detergent-resistant membranes (DRMs). DRM association required acid activation of the purified toxin prior to contact with cells, and acid activation also was required for VacA cytotoxicity. Treatment of cells with methyl-beta-cyclodextrin (a cholesterol-depleting agent) did not inhibit VacA-induced depolarization of the plasma membrane, but interfered with the internalization or intracellular localization of VacA and inhibited the capacity of the toxin to induce cell vacuolation. Treatment of cells with nystatin also inhibited VacA-induced cell vacuolation. These data indicate that VacA associates with lipid raft microdomains in the absence of GPI-anchored proteins and suggest that association of the toxin with lipid rafts is important for VacA cytotoxicity.     

Sphingomyelin functions as a novel receptor for Helicobacter pylori VacA

The vacuolating cytotoxin (VacA) of the gastric pathogen Helicobacter pylori binds and enters epithelial cells, ultimately resulting in cellular vacuolation. Several host factors have been reported to be important for VacA function, but none of these have been demonstrated to be essential for toxin binding to the plasma membrane. Thus, the identity of cell surface receptors critical for both toxin binding and function has remained elusive. Here, we identify VacA as the first bacterial virulence factor that exploits the important plasma membrane sphingolipid, sphingomyelin (SM), as a cellular receptor. Depletion of plasma membrane SM with sphingomyelinase inhibited VacA-mediated vacuolation and significantly reduced the sensitivity of HeLa cells, as well as several other cell lines, to VacA. Further analysis revealed that SM is critical for VacA interactions with the plasma membrane. Restoring plasma membrane SM in cells previously depleted of SM was sufficient to rescue both toxin vacuolation activity and plasma membrane binding. VacA association with detergent-resistant membranes was inhibited in cells pretreated with SMase C, indicating the importance of SM for VacA association with lipid raft microdomains. Finally, VacA bound to SM in an in vitro ELISA assay in a manner competitively inhibited by lysenin, a known SM-binding protein. Our results suggest a model where VacA may exploit the capacity of SM to preferentially partition into lipid rafts in order to access the raft-associated cellular machinery previously shown to be required for toxin entry into host cells.     

Helicobacter pylori vacuolating cytotoxin, VacA, is responsible for gastric ulceration

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Most H. pylori strains secrete VacA into the extracellular space. After exposure of VacA to acidic or basic pH, re-oligomerized VacA (mainly 6 monomeric units) at neutral pH is more toxic. Although the mechanisms have not been defined, VacA induces multiple effects on epithelial and lymphatic cells, i.e., vacuolation with alterations of endo-lysosomal function, anion-selective channel formation, mitochondrial damage, and the inhibition of primary human CD4+ cell proliferation. VacA binds to two types of receptor-like protein tyrosine phosphatases (RPTP), RPTPalpha and RPTPbeta, on the surface of target cells. Oral administration of VacA to wild-type mice, but not to RPTPbeta KO mice, results in gastric ulcers, suggesting that RPTPbeta is essential for intoxication of gastric tissue by VacA. As the potential roles of VacA as a ligand for RPTPalpha and RPTPbeta are only poor understood, further studies are needed to determine the importance of VacA in the pathogenisis of disease due to H. pylori infection.     

Brefeldin A enhances Helicobacter pylori vacuolating cytotoxin-induced vacuolation of epithelial cells

Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region. We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA. The effect is BFA dose-dependent and occurs within 2.5 h. These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently. This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo.     

Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.     

Plant polyphenols inhibit VacA,a toxin secreted by the gastric pathogen Helicobacter pylori

VacA is a major virulence factor of the widespread stomach-dwelling bacterium Helicobacter pylori. It causes cell vacuolation and tissue damage by forming anion-selective, urea-permeable channels in plasma and endosomal membranes. We report that several flavone derivatives and other polyphenols present in vegetables and plants inhibit ion and urea conduction and cell vacuolation by VacA. Red wine and green tea, which contain many of the compounds in question, also potently inhibit the toxin. These observations suggest that polyphenols or polyphenol derivatives may be useful in the prevention or cure of H. pylori-associated gastric diseases.     

Helicobacter pylori toxin VacA is transferred to host cells via a novel contact-dependent mechanism

Helicobacter pylori is the causative agent of peptic ulcer disease. A major virulence factor of H. pylori is VacA, a toxin that causes massive vacuolization of epithelial cell lines in vitro and gastric epithelial erosion in vivo. Although VacA is exported over the outer membrane and is released from the bacteria, a portion of the toxin remains associated with the bacterial surface. We have found surface-associated toxin to be biologically active and spatially organized into distinct toxin-rich domains on the bacterial surface. Upon bacterial contact with host cells, toxin clusters are transferred directly from the bacterial surface to the host cell surface at the bacteria-cell interface, followed by uptake and intoxication. This contact-dependent transfer of VacA represents a cost-efficient route for delivery of VacA and potentially other bacterial effector molecules to target cells.     

Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: possible implications for the mechanism of cellular vacuolation

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.     

Early endosomes associated with dynamic F-actin structures are required for late trafficking of H. pylori VacA toxin

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed by a clathrin- independent pathway into vesicles named GPI-AP-enriched early endosomal compartments (GEECs). We recently showed that the vacuolating toxin VacA secreted by Helicobacter pylori is endocytosed into the GEECs (Gauthier, N.C., P. Monzo, V. Kaddai, A. Doye, V. Ricci, and P. Boquet. 2005. Mol. Biol. Cell. 16:4852-4866). Unlike GPI-APs that are mostly recycled back to the plasma membrane, VacA reaches early endosomes (EEs) and then late endosomes (LEs), where vacuolation occurs. In this study, we used VacA to study the trafficking pathway between GEECs and LEs. We found that VacA routing from GEECs to LEs required polymerized actin. During this trafficking, VacA was transferred from GEECs to EEs associated with polymerized actin structures. The CD2-associated protein (CD2AP), a docking protein implicated in intracellular trafficking, bridged the filamentous actin (F-actin) structures with EEs containing VacA. CD2AP regulated those F-actin structures and was required to transfer VacA from GEECs to LEs. These results demonstrate that sorting from GEECs to LEs requires dynamic F-actin structures on EEs.     

A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1 Helicobacter pylori VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation

Helicobacter pylori, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of H. pylori vacA alleles can be distinguished based on sequence diversity in the "middle" region (i.e., m1 and m2) and in the 5' end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from H. pylori strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P(Cl)/P(Na) = 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Delta6-27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Delta6-27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many H. pylori isolates suggests that type s2 VacA proteins retain an important biological activity.     

Expression and binding analysis of GST-VacA fusions reveals that the C-terminal approximately 100-residue segment of exotoxin is crucial for binding in HeLa cells

The Helicobacter pylori toxin VacA induces intracellular vacuolation and plays an essential role in H. pylori-related diseases. The mature exotoxin is divided into two domains, P37 and P58. A soluble form of VacA fused with GST was expressed in Escherichia coli. Although the soluble fusion lacked vacuolating activity after cleavage by thrombin, it had a binding affinity similar to that of the native VacA. Moreover, it blocked the vacuolating activity induced by the native toxin. Different C-terminal truncated fusions were generated (GST-P72, GST-P53, and GST-P37) and were also produced in a soluble form. A significantly reduced binding activity was seen for GST-P72 and nearly no specific association was detected for GST-P37. Our results suggested that the whole P58 fragment contributed to the cell binding activity in HeLa cells, particularly in the C-terminal approximately 100-residue region.