Functional complementation reveals the importance of intermolecular monomer interactions for Helicobacter pylori VacA vacuolating activity

The Helicobacter pylori vacuolating cytotoxin (VacA) induces degenerative vacuolation of sensitive mammalian cell lines. Although evidence is accumulating that VacA enters cells and functions from an intracellular site of action, the biochemical mechanism by which VacA mediates cellular vacuolation has not been established. In this study, we used functional complementation and biochemical approaches to probe the structure of VacA. VacA consists of two discrete fragments, p37 and p58, that are both required for vacuolating activity. Using a transient transfection system, we expressed genetically modified forms of VacA and identified mutations in either p37 or p58 that inactivated the toxin. VacA with an inactivating single-residue substitution in the p37 domain [VacA (P9A)] functionally complemented a second mutant form of VacA with an inactivating two-residue deletion in the p58 domain [VacA Delta(346-347)]. VacA (P9A) and VacA Delta(346-347) also co-immunoprecipitated from vacuolated monolayers, supporting the hypothesis that these two inactive mutants associate directly to function in trans. p37 and p58 interact directly when expressed as separate fragments within HeLa cells, suggesting that p37-p58 inter-actions facilitate VacA monomer associations. Collectively, these results support a model in which the active form of VacA requires assembly into a complex of two or more monomers to elaborate toxin function.     

Two distinctive cell binding patterns by vacuolating toxin fused with glutathione S-transferase: one high-affinity m1-specific binding and the other lower-affinity binding for variant m forms

The Helicobacter pylori VacA causes large intracellular vacuoles in epithelial cells such as HeLa or RK13 cells. Two major VacA forms, m1 and m2, divergent in an approximately 300 amino acid segment within the cell binding domain P58, display distinct cell-type specificity. Sequence analysis of four vacA alleles showed that a m1-like allele (61) and two m2 alleles (62 and v226) mainly differed in the midregion and that v225, a m1m2 chimera, was a natural double crossover from v226 and another allele. Each of these alleles was expressed as a soluble GST-VacA fusion that did not form a large oligomer. The recombinant VacA portion nevertheless assembled into higher ordered structures and possessed biological binding activity similar to that of the native VacA. A direct comparison of fusion-cell binding activity showed that m1 > m1m2 > m2 in HeLa cells, whereas there were more similar activities in RK13 cells. Vacuolating analyses of three forms revealed a positive correlation between cell binding activity and vacuolating activity. Moreover, the m1-type N-terminal half portion of the midregion was crucial for HeLa cell cytotoxicity. Kinetic, Scatchard, and inhibition analyses suggested the presence of at least two receptors: a m1-type specific high-affinity receptor (K(d) = approximately 5 nM) and a common VacA receptor interacting similarly with m1, m1m2, and m2 via a lower affinity (K(d) = 45-67 nM). Expression of mainly the m1-type receptor on HeLa cells whereas both receptors on RK13 cells may account for distinct cell binding activity and therefore for cell-type specificity.     

Human gastric glycosphingolipids recognized by Helicobacter pylori vacuolating cytotoxin VacA

Many bacterial toxins utilize cell surface glycoconjugate receptors for attachment to target cells. In the present study the potential carbohydrate binding of Helicobacter pylori vacuolating cytotoxin VacA was investigated by binding to human gastric glycosphingolipids on thin-layer chromatograms. Thereby a distinct binding of the toxin to two compounds in the non-acid glycosphingolipid fraction was detected. The VacA-binding glycosphingolipids were isolated and characterized by mass spectrometry and proton NMR as galactosylceramide (Galbeta1Cer) and galabiosylceramide (Galalpha4Galbeta1Cer). Comparison of the binding preferences of the protein to reference glycosphingolipids from other sources showed an additional recognition of glucosylceramide (Glcbeta1Cer), lactosylceramide (Galbeta4Glcbeta1Cer) and globotriaosylceramide (Galalpha4Galbeta4Glcbeta1Cer). No binding to the glycosphingolipids recognized by the VacA holotoxin was obtained with a mutant toxin with deletion of the 37 kDa fragment of VacA (P58 molecule). Collectively our data show that the VacA cytotoxin is a glycosphingolipid binding protein, where the 37 kDa moiety is required for carbohydrate recognition. The ability to bind to short chain glycosphingolipids will position the toxin close to the cell membrane, which may facilitate toxin internalization.     

Helicobacter pylori vacuolating cytotoxin binding to a putative cell surface receptor, heparan sulfate, studied by surface plasmon resonance

The Helicobacter pylori vacuolating cytotoxin or VacA toxin is a major virulence factor in H. pylori infection and type B gastritis. We predicted heparin/heparan sulfate (H/HS) binding properties of the 58-kDa subunit of VacA cytotoxin using bioinformatics tools and showed this by surface plasmon resonance (SPR)-based biosensor studies. Putative H/HS binding peptides were synthesized and binding to HS was shown by SPR in the absence or presence of trifluoroethanol. We found that a recombinant cytotoxin VacA polypeptide binds to surface-immobilized HS and propose that HS might be a receptor/co-receptor for H. pylori VacA cytotoxin.     

Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin

Disease-associated strains of Helicobacter pylori produce a potent toxin that is believed to play a key role in peptic ulcer disease in man. In vitro the toxin causes severe vacuolar degeneration in target cells and has thus been termed VacA (for vacuolating cytotoxin A). Cytotoxic activity is associated with a > 600-kD protein consisting of several copies of a 95-kD polypeptide that undergoes specific proteolytic cleavage after release from the bacteria to produce 37- and 58-kD fragments. Quick freeze, deep etch electron microscopy has revealed that the native cytotoxin is formed as regular oligomers with either six- or seven-fold radial symmetry. Within each monomer, two domains can clearly be distinguished, suggesting that the 37- and 58-kD fragments derive from proteolytic cleavage between discrete subunits of the monomer. Analysis of preparations of the toxin that had undergone extensive cleavage into the 37- and 58-kD subunits supports this interpretation and reveals that after cleavage the subunits remain associated in the oligomeric structure. The data suggest a structural similarity with AB-type toxins.     

Functional properties of the p33 and p55 domains of the Helicobacter pylori vacuolating cytotoxin

Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties.     

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.     

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.     

Identification of the minimal intracellular vacuolating domain of the Helicobacter pylori vacuolating toxin

Helicobacter pylori secretes a cytotoxin (VacA) that induces the formation of large vacuoles originating from late endocytic vesicles in sensitive mammalian cells. Although evidence is accumulating that VacA is an A-B toxin, distinct A and B fragments have not been identified. To localize the putative catalytic A-fragment, we transfected HeLa cells with plasmids encoding truncated forms of VacA fused to green fluorescence protein. By analyzing truncated VacA fragments for intracellular vacuolating activity, we reduced the minimal functional domain to the amino-terminal 422 residues of VacA, which is less than one-half of the full-length protein (953 amino acids). VacA is frequently isolated as a proteolytically nicked protein of two fragments that remain noncovalently associated and retain vacuolating activity. Neither the amino-terminal 311 residue fragment (p33) nor the carboxyl-terminal 642 residue fragment (p70) of proteolytically nicked VacA are able to induce cellular vacuolation by themselves. However, co-transfection of HeLa cells with separate plasmids expressing both p33 and p70 resulted in vacuolated cells. Further analysis revealed that a minimal fragment comprising just residues 312-478 functionally complemented p33. Collectively, our results suggest a novel molecular architecture for VacA, with cytosolic localization of both fragments of nicked toxin required to mediate intracellular vacuolating 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.     

Structural organization of membrane‐inserted hexamers formed by Helicobacter pylori VacA toxin

Helicobacter pylori colonizes the human stomach and is a potential cause of peptic ulceration or gastric adenocarcinoma. H. pylori secretes a pore‐forming toxin known as vacuolating cytotoxin A (VacA). The 88 kDa secreted VacA protein, composed of an N‐terminal p33 domain and a C‐terminal p55 domain, assembles into water‐soluble oligomers. The structural organization of membrane‐bound VacA has not been characterized in any detail and the role(s) of specific VacA domains in membrane binding and insertion are unclear. We show that membrane‐bound VacA organizes into hexameric oligomers. Comparison of the two‐dimensional averages of membrane‐bound and soluble VacA hexamers generated using single particle electron microscopy reveals a structural difference in the central region of the oligomers (corresponding to the p33 domain), suggesting that membrane association triggers a structural change in the p33 domain. Analyses of the isolated p55 domain and VacA variants demonstrate that while the p55 domain can bind membranes, the p33 domain is required for membrane insertion. Surprisingly, neither VacA oligomerization nor the presence of putative transmembrane GXXXG repeats in the p33 domain is required for membrane insertion. These findings provide new insights into the process by which VacA binds and inserts into the lipid bilayer to form membrane channels.     

Interactions between p-33 and p-55 domains of the Helicobacter pylori vacuolating cytotoxin (VacA)

The VacA toxin secreted by Helicobacter pylori is considered to be an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. VacA monomers self-assemble into water-soluble oligomeric structures and can form anion-selective membrane channels. The goal of this study was to characterize VacA-VacA interactions that may mediate assembly of VacA monomers into higher order structures. We investigated potential interactions between two domains of VacA (termed p-33 and p-55) by using a yeast two-hybrid system. p-33/p-55 interactions were detected in this system, whereas p-33/p-33 and p-55/p-55 interactions were not detected. Several p-33 proteins containing internal deletion mutations were unable to interact with wild-type p-55 in the yeast two-hybrid system. Introduction of these same deletion mutations into the H. pylori vacA gene resulted in secretion of mutant VacA proteins that failed to assemble into large oligomeric structures and that lacked vacuolating toxic activity for HeLa cells. Additional mapping studies in the yeast two-hybrid system indicated that only the N-terminal portion of the p-55 domain is required for p-33/p-55 interactions. To characterize further p-33/p-55 interactions, we engineered an H. pylori strain that produced a VacA toxin containing an enterokinase cleavage site located between the p-33 and p-55 domains. Enterokinase treatment resulted in complete proteolysis of VacA into p-33 and p-55 domains, which remained physically associated within oligomeric structures and retained vacuolating cytotoxin activity. These results provide evidence that interactions between p-33 and p-55 domains play an important role in VacA assembly into oligomeric structures.     

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.     

Sticky socks: Helicobacter pylori VacA takes shape

Several receptors have been described for the Helicobacter pylori vacuolating toxin VacA, which exerts different effects on epithelial cells and on immune cells. The crystal structure of the putative receptor-binding domain of VacA (p55) has now been solved. It consists of a parallel beta-helix with a C-terminal globular domain. A comparison between allelic variants of p55 and docking of the p55 domain into the quaternary structure, as shown by electron microscopy, revealed structural features that might be important for elucidating the molecular details of receptor interaction and channel formation.     

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.     

Helicobacter pylori vacuolating cytotoxin enters cells, localizes to the mitochondria, and induces mitochondrial membrane permeability changes correlated to toxin channel activity

The Helicobacter pylori vacuolating cytotoxin (VacA) intoxicates mammalian cells resulting in reduction of mitochondrial transmembrane potential (Delta Psi m reduction) and cytochrome c release, two events consistent with the modulation of mitochondrial membrane permeability. We now demonstrate that the entry of VacA into cells and the capacity of VacA to form anion-selective channels are both essential for Delta Psi m reduction and cytochrome c release. Subsequent to cell entry, a substantial fraction of VacA localizes to the mitochondria. Neither Delta Psi m reduction nor cytochrome c release within VacA-intoxicated cells requires cellular caspase activity. Moreover, VacA cellular activity is not sensitive to cyclosporin A, suggesting that VacA does not induce the mitochondrial permeability transition as a mechanism for Delta Psi m reduction and cytochrome c release. Time-course and dose-response studies indicate that Delta Psi m reduction occurs substantially before and at lower concentrations of VacA than cytochrome c release. Collectively, these results support a model that VacA enters mammalian cells, localizes to the mitochondria, and modulates mitochondrial membrane permeability by a mechanism dependent on toxin channel activity ultimately resulting in cytochrome c release. This model represents a novel mechanism for regulation of a mitochondrial-dependent apoptosis pathway by a bacterial toxin.     

High cell sensitivity to Helicobacter pylori VacA toxin depends on a GPI-anchored protein and is not blocked by inhibition of the clathrin-mediated pathway of endocytosis

Helicobacter pylori vacuolating toxin (VacA) causes vacuolation in a variety of cultured cell lines, sensitivity to VacA differing greatly, however, among the different cell types. We found that the high sensitivity of HEp-2 cells to VacA was impaired by treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) which removes glycosylphosphatidylinositol (GPI)-anchored proteins from the cell surface. Incubation of cells with a cholesterol-sequestering agent, that impairs both structure and function of sphingolipid-cholesterol-rich membrane microdomains ("lipid rafts"), also impaired VacA-induced cell vacuolation. Overexpression into HEp-2 cells of proteins inhibiting clathrin-dependent endocytosis (i.e., a dominant-negative mutant of Eps15, the five tandem Src-homology-3 domains of intersectin, and the K44A dominant-negative mutant of dynamin II) did not affect vacuolation induced by VacA. Nevertheless, F-actin depolymerization, known to block the different types of endocytic mechanisms, strongly impaired VacA vacuolating activity. Taken together, our data suggest that the high cell sensitivity to VacA depends on the presence of one or several GPI-anchored protein(s), intact membrane lipid rafts, and an uptake mechanism via a clathrin-independent endocytic pathway.     

Inhibition of the vacuolating and anion channel activities of the VacA toxin of Helicobacter pylori.

VacA, the vacuolating cytotoxin secreted by Helicobacter pylori, is believed to be a major causative factor in the development of gastroduodenal ulcers. This toxin causes vacuolation of cultured cells and it has recently been found to form anion-selective channels upon insertion into planar bilayers as well as in the plasma membrane of HeLa cells. Here, we identify a series of inhibitors of VacA channels and we compare their effectiveness as channel blockers and as inhibitors of VacA-induced vacuolation, confirming that the two phenomena are linked. This characterization opens the way to studies in other experimental systems and to the search for a specific inhibitor of VacA action.     

Glycosylphosphatidylinositol-anchored proteins and actin cytoskeleton modulate chloride transport by channels formed by the Helicobacter pylori vacuolating cytotoxin VacA in HeLa cells

The vacuolating cytotoxin VacA is an important virulence factor of Helicobacter pylori. Removing glycosylphosphatidylinositol-anchored proteins (GPI-Ps) from the cell surface by phosphatidylinositol-phospholipase C or disrupting the cell actin cytoskeleton by cytochalasin D reduced VacA-induced vacuolation of cells. Using the fluorescent dye 6-methoxy-N-ethylquinolinium chloride, an indicator for cytosolic chloride, we have investigated the role of either GPI-Ps or actin cytoskeleton in the activity of the selective anionic channel formed by VacA at the plasma membrane level. Removal of GPI-Ps from HeLa cell surfaces did not impair VacA localization into lipid rafts but strongly reduced VacA channel-mediated cell influx and efflux of chloride. Disruption of the actin cytoskeleton of HeLa cells by cytochalasin D did not affect VacA localization in lipid rafts but blocked VacA cell internalization and inhibited cell vacuolation while increasing the overall chloride transport by the toxin channel at the cell surface. Specific enlargement of Rab7-positive compartments induced by VacA could be mimicked by the weak base chloroquine alone, and the vacuolating activities of either chloroquine alone or VacA were blocked with the same potency by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid shown to inhibit VacA channel activity. We suggest that formation of functional VacA channels at the cell surface required GPI-Ps and that endocytosis of these channels by an actin-dependent process increases the chloride content of late endosomes that accumulate weak bases, provoking their enlargement by osmotic swelling.     

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.