The vacA Gene

All H. pylori strains contain a copy of the toxin gene, vacA. The vacA transcript is monocistronic, with its transcriptional start point located about 119 nucleotides upstream from the ATG start codon (28, 29, 90). Insertional mutagenesis of vacA abrogates the capacity of H. pylori to induce vacuolation in epithelial cells, and ablates several other vacA-induced toxic effects (13, 78, 90). Alleles of vacA from at least 25 different H. pylori strains have been sequenced and range from 3,864 to 3,933 nucleotides in length (2, 13, 44, 68, 80, 90, 103). As discussed later in this chapter, there is considerable genetic diversity among vacA alleles from different strains, and alleles can be categorized into several families. The most extensively studied form of VacA is encoded by type s1/m1 vacA alleles, which typically encode VacA proteins associated with a high level of vacuolating cytotoxin activity (Fig. 1) (2). Other forms of VacA are associated with lower level or absent vacuolating activity. For simplicity, we will initially describe the characteristics of prototypic s1/m1 forms of VacA.

Figure 1

Schematic of the vacuolating cytotoxin gene, vacA, and protein, VacA (not to scale). vacA alleles vary between strains and this variation is most marked in the second part of the signal region and the ~800-bp mid region. Alleles can be classified according (more...)

The VacA Protein

Processing and secretion of VacA

VacA is predicted to encode a protoxin with a mass of about 140 kDa, but the mature secreted VacA toxin migrates as a band of approximately 90 kDa under denaturing conditions (10, 13, 80, 90, 103). A comparison of the amino-terminal sequence of the mature secreted toxin with that predicted for the protoxin indicates that a 33-amino-acid amino-terminal signal sequence is cleaved during the process of VacA secretion (Fig. 1). Studies using antisera raised against different regions of recombinant VacA show that a polypeptide of about 33 kDa derived from the carboxy-terminal portion of the protoxin remains localized to the bacteria and is not secreted (Fig. 1) (103). This carboxy-terminal portion of VacA is predicted to contain amphipathic β-sheets capable of forming a β-barrel structure and has a terminal phenylalanine-containing motif that is present in many outer membrane proteins (90). These features, together with a pair of cysteine residues near the carboxy terminus of the mature secreted protein, are characteristic of a family of secreted bacterial proteins called autotransporters (42). Autotransporters do not require any ancillary proteins for export across the bacterial outer membrane. Our current understanding of autotransporter export is based primarily on studies of Neisseria gonorrhoeae IgA1 protease. Translocation of IgA1 protease across the bacterial cytoplasmic membrane is accomplished via a Sec-mediated process and is accompanied by cleavage of an amino-terminal signal peptide. The carboxy-terminal β-barrel domain then inserts into the outer membrane and is thought to function as a pore through which the rest of the molecule passes. Autoproteolytic cleavage yields the mature secreted IgA1 protease; the carboxy-terminal domain remains associated with the outer membrane (81).

Oligomeric structure of VacA

Early studies indicated that, although mature VacA monomers are approximately 90 kDa in mass, the toxin exists as a much larger complex or aggregate under nondenaturing conditions (10). Lupetti et al. examined the ultrastructure of purified VacA using deep-etch electron microscopy and demonstrated that the toxin assembles into large flower-shaped complexes, about 30 nm in diameter, which appear to consist of a central ring surrounded by six or seven "petals" (54). Three-dimensional reconstructions of these deep-etch metal replicas have provided a detailed view of the surface of VacA oligomers (Fig. 2) (50). As well as the classical flower-like complexes, VacA can assemble into a different type of complex, termed a "flat form," which consists of six or seven petals without a prominent central ring (11, 54). The petals that comprise the flat form typically radiate from the center of the complex with a distinctive clockwise chirality. Multiple models have been proposed to explain the assembly of VacA into flowerlike complexes and chiral flat forms. In one model, the flower-like forms are suggested to comprise six or seven monomers of about 90 kDa (50, 54). In another model, the flower-like forms are suggested to be dodecamers or tetradecamers of VacA monomers of about 90 kDa, and flat forms are suggested to be hexamers or heptamers (11). When VacA is exposed to acidic or alkaline pH, VacA oligomers dissociate into monomeric components of about 90 kDa, each measuring about 6 by 14 nm (11, 59, 117). This pH-mediated disassembly is associated with a marked increase in VacA cytotoxic activity (11, 21, 58, 117). It is currently thought that water-soluble VacA oligomers possess relatively little cytotoxic activity compared to VacA monomers.

Figure 2

Image of the top surface of a soluble VacA oligomer, which has a diameter of approximately 30 nm. This is a three-dimensional reconstruction from electron micrographs of quick-freeze deep-etch metal replicas of VacA. (Reprinted from reference with permission.) (more...)

Further investigation of VacA structure has been undertaken using atomic force microscopic imaging of purified toxin bound to supported lipid bilayers (Fig. 3) (15). Isolated VacA oligomers imaged by this approach appear similar to the structures imaged by deep-etch electron microscopy. Two-dimensional crystalline arrays of VacA on lipid bilayers consist of an ordered array of hexagonal central rings attached by thin connectors to peripheral domains (Fig. 3).

Figure 3

(A) Atomic force microscopy image of VacA two-dimensional crystals on a supported lipid bilayer. (B) Processed image of VacA two-dimensional crystals. The line diagram in the top right corner illustrates the orientation of a single oligomer within the (more...)

H. pylori mutant strains, constructed with in-frame deletions in the portion of vacA encoding the amino-terminal region of the toxin, express truncated VacA proteins that are secreted but fail to oligomerize and lack detectable cytotoxic activity (83, 111). One of these mutant VacA proteins (VacA Δ91–330) has been characterized in detail, and forms water-soluble dimers that have an ultrastructural appearance similar to that of the peripheral petals of VacA oligomers (83). This suggests that the peripheral petals of VacA oligomers correspond to the carboxy-terminal portion of the mature secreted VacA polypeptide, most likely the 58-kDa domain described below.

Identification of functional domains in VacA

Limited proteolysis yields two VacA domains

During prolonged storage or upon incubation with trypsin (11, 54, 103) the purified ~90-kDa VacA toxin tends to degrade into ~37- and ~58-kDa components, which are derived from the amino terminus and carboxy terminus of the protein, respectively (Fig. 1). Proteolytic cleavage occurs at a site containing multiple charged amino acids, which are predicted to be surface exposed (103). The 37- and 58-kDa fragments of VacA have been presumed to represent subunits or domains of the holotoxin. Recent mass spectrometry data indicate that the two VacA fragments have masses of 33 and 55 kDa (66), but in this chapter we will continue to refer to the fragments as 37 and 58 kDa to avoid confusion with the published literature. To determine whether cleavage of VacA into 37- and 58-kDa fragments is required for toxin activity, Burroni et al. constructed an H. pylori mutant in which the region of vacA encoding the 46 amino acids flanking the VacA cleavage site was deleted (8). This mutant VacA was fully active, indicating that cleavage of the exposed loop is not necessary for activity. Interestingly, however, in contrast to the wild-type VacA produced by the parent strain, which preferentially formed seven-sided complexes, the mutant preferentially formed six-sided complexes, indicating that removing the exposed loop had introduced structural constraints.

Activity of the amino-terminal portion of VacA in the cell cytosol

The minimal region of VacA required for vacuolating activity has been defined in experiments where mutant forms of vacA under the control of a eukaryotic promoter have been expressed from plasmids in the cytosol of epithelial cells (17). In these experiments, epithelial cell lines were transfected with plasmid constructs encoding either the full-length ~90-kDa secreted toxin or amino- or carboxy-terminally truncated fragments. These experiments showed that a VacA protein lacking most of the carboxy-terminal 58-kDa domain retained full vacuolating activity (18, 121). However, activity was completely abrogated by removing 10 amino acids from the amino-terminus and partially abrogated by removing 6 amino acids (18, 121). Thus, the minimal VacA domain that exhibited full vacuolating activity when expressed intracellularly was a peptide comprising amino acids 1–422, that is, the 37-kDa domain plus a fragment of the 58-kDa domain (121). Interestingly, coexpression of the 37-kDa fragment, which alone was inactive, with a fragment containing the amino-terminal 165 amino acids of the 58-kDa fragment resulted in full vacuolating activity (121). Hydrophobicity plots reveal a possible explanation for the importance of the VacA amino terminus: part of this region (amino acids 1–32) is the only hydrophobic region in VacA long enough to span a membrane. To investigate this further, an H. pylori vacA partial deletion mutant was constructed that lacked codons for amino acids 6–27 (114). This mutant did not exhibit any obvious alterations in structure compared to wild-type VacA but lacked cytotoxic activity. Furthermore, alanine scanning mutagenesis revealed that point mutations at proline 9 or glycine 14 completely abolished VacA activity (120). Finally, addition of an amino-terminal hydrophilic extension to VacA (identical to that found in the naturally occurring nontoxigenic s2 forms of VacA discussed later) abolished toxin activity (51). Together, these observations support a critical role for the amino-terminal hydrophobic region in toxin activity.

Localization of a receptor binding region

Several lines of evidence indicate that binding of VacA to cells is mediated by amino acid sequences located in the carboxy-terminal portion of the mature protein (corresponding to the 58-kDa domain). First, studies with the purified 58-kDa fragment from a mutant H. pylori strain indicate that this protein binds to HeLa cells with kinetics similar to those of the intact toxin (83). Second, polyclonal antiserum reactive with the 58-kDa domain inhibits the binding of VacA to cells (31). Third, some naturally occurring forms of VacA, which have markedly divergent amino acid sequences in the 58-kDa domain (called m2 forms, and discussed further below), cause vacuolation in a more restricted range of cultured epithelial cell lines than the m1 forms of VacA discussed thus far, one explanation for which would be differences in cell binding (68). Finally, VacA with a type m2 58-kDa domain, which did not cause HeLa cell vacuolation when applied externally, caused vacuolation when expressed from a plasmid in the HeLa cell cytoplasm. This implies that m2 VacA is fully active but cannot get to its site of action; one explanation of this would be inability to bind to the cell (17). Experiments using naturally occurring and engineered m1/m2 chimeric proteins (46) suggest that an ~40 amino acid region near the amino-terminal end of the 58-kDa domain is necessary for HeLa cell vacuolation and may be involved in HeLa cell binding.

Biological Activity of VacA (Fig. 4)

Cytotoxicity

VacA causes epithelial cell vacuolation in vitro, but this does not lead rapidly to cell death. For primary human gastric epithelial cells exposed to high doses of toxin, cell death has been documented after 2 days (95). In contrast, cell death does not usually occur in immortalized cell lines exposed to the toxin; for example, incubation of AZ-521 gastric epithelial cells with VacA for several hours causes reduced mitochondrial ATP production and reduced oxygen consumption but does not result in cell death (48). Whether VacA causes epithelial cell death in vivo in humans is unknown, but in mice oral administration of VacA leads to erosion of the gastric epithelium, presumably involving cellular loss (107).

VacA binding

The binding and uptake of VacA by cells are not yet clearly understood, and there are many apparent contradictions in this area of research. The prototypic s1/m1 form of VacA binds to HeLa cells in a saturable manner when assessed by flow-cytometry analysis (57). In contrast, saturable binding has not been detected with classical ligand binding assays with ¹²I-labeled VacA (58). Activation of VacA by acid treatment markedly enhances its vacuolating activity but does not significantly increase its binding to HeLa or BHK cells (57–59). In contrast, acid activation enhances binding of the toxin to the gastric cell line AZ-521 (117). Several specific VacA receptors have been proposed. In the AZ-521 system, activated VacA binds to a 250-kDa receptor protein-tyrosine phosphatase β (RPTPβ), which regulates intracellular tyrosine phosphorylation (67, 117). Several lines of evidence suggest a vital role for RPTPβ in binding of VacA to cells and subsequent intoxication. First, treatment of the HL-60 cell line with phorbol 12-myristate 13-acetate (PMA) leads to induction of RPTPβ expression, which is accompanied by induction of VacA sensitivity (20). Second, BHK-21 cells, which are insensitive to VacA, can be made sensitive by transfection with expression vectors containing the RPTPβ gene. Finally, ablation of RPTPβ synthesis by antisense oligonucleotides in PMA-treated HL-60 cells results in a significant decrease in VacA-induced vacuolation. Two other specific VacA receptors have been reported: an unidentified 140-kDa protein in AZ-521 and AGS cells (116) and the epidermal growth factor receptor in HeLa cells (91). Taken together, these observations suggest the existence of multiple surface-binding sites recognized by both inactive and activated VacA and also the presence of specific VacA receptors that are variably expressed in different cell lines.

Figure 4

Cartoon illustrating a possible model for interaction of VacA with the epithelial cell. The inactive, soluble oligomer, made up of p37 and p58 fragments, can be disrupted by acidic or alkaline treatment to release active monomeric VacA. Binding: Both (more...)

VacA internalization and intracellular trafficking

Both 58-kDa and 37-kDa regions are required for VacA internalization (83). To be internalized, VacA must be preactivated by exposure to acid or alkali (58). Internalization occurs through an energy-dependent process, the precise nature of which is unclear but which may be receptor-mediated endocytosis. After internalization, VacA molecules localize in membrane vesicles (31) and are transported along the endocytic pathway to vacuolar-type (V-) ATPase-positive late endosomes and lysosomes, where they accumulate and persist for days with little evidence of degradation (85, 96).

Cell vacuolation and impairment of endolysosmal function

The induction of intracellular vacuoles was the first characterized action of VacA (14, 53). The vacuolar membranes contain both late endosomal and lysosomal markers, suggesting that the vacuoles are derived from these compartments (62, 75). The formation of VacA-induced vacuoles requires the full activity of V-ATPase and the presence of weak bases (12, 14, 74, 85), suggesting that vacuoles are derived from the accumulation of weak bases within acidic compartments followed by water influx and swelling. In addition, two small GTP-binding proteins have been shown to be involved in vacuole biogenesis: the membrane traffic regulator rab7 and the actin-cytoskeleton-associated Rac1 (43, 76). These two proteins are associated with the membrane of VacA-induced vacuoles. Vacuolation is inhibited by the expression of rab7 or Rac1 dominant negative mutants and is potentiated by dominant positive mutants. These observations suggest that vacuole development is regulated by membrane fusion events and by the cytoskeleton supporting late endosomal compartments. In HeLa cells, VacA impairs the transport of acidic hydrolases to lysosomes, resulting in release of these enzymes into the extracellular medium (89). Moreover, the degradative power of HeLa cell lysosomes (89) and of the antigen-processing compartment of B lymphocytes (61) is also reduced by VacA. Such early functional alteration of the endocytic pathway, occurring in the absence of vacuolation, is likely to be due to a partial neutralization of acidic compartments (89).

Alteration of epithelial permeability

Epithelial monolayers of MDCK I, T84, or epH4 cells on porous filters are not vacuolated by VacA and do not show signs of endolysosomal dysfunction (77). However, following exposure to VacA, transepithelial electrical resistance (TER) decreases, accompanied by an increase in transepithelial flux of low-molecular-weight molecules (77). The size selectivity of this increased epithelial permeation, the lack of accompanying vacuolation, and the lack of redistribution of junctional proteins all suggest that VacA modulates the resistance of these model epithelia through a paracellular effect. Only epithelial cell monolayers able to develop a TER higher than 1,000 to 1,200 Ω/cm² are affected. The use of isogenic mutant strains confirms that the effect is dependent on VacA (78). The naturally occurring m2 type of VacA discussed later reduces TER in MDCK cells but does not cause vacuolation in this cell line even when cells are nonconfluent (78), further confirming that vacuolation and increased permeability of monolayers are discrete and independent effects.

Ion channel formation

VacA forms ion channels in model lipid bilayers and cell plasma membranes, and this phenomenon may underlie all the other effects of VacA. Disassembly of the inactive VacA oligomer by acidic conditions allows insertion of the toxin into lipid bilayers (61, 62, 69). Experiments with planar model membranes show that membrane insertion is followed by the formation of voltage-dependent, low-conductance (10 to 30 pS in 2M KCl), anion-selective channels (45, 104). Patch clamp analysis of HeLa cells demonstrates that VacA forms plasma membrane channels with properties similar to those observed in model membranes (100). Various anion channel blockers inhibit VacA channels in vitro with different potencies and are able to prevent and partially reverse vacuolation of HeLa cells (100, 105), indicating an essential role of the anion channel in vacuolation. The development of a dominant negative mutant of VacA, able to form mixed oligomers with wild-type VacA, provides additional evidence suggesting that functional anion channel formation is required for cell vacuolation (117). It has been proposed that the endocytosed VacA channel, by allowing anions to permeate into late endosomes, increases the turnover of the electrogenic V-ATPase, which leads to accumulation of weak bases (when present) and thence to vacuole formation by water influx (102, 108). This hypothesis is in keeping with the observation that internalization of surface-bound VacA is necessary for the subsequent development of vacuolation (58). In this model, vacuolation can be thought of as a side effect of the massive accumulation of endocytosed VacA channels in endolysosomes.

VacA epithelial permeabilization of MDCK I cells can be partially prevented and reversed by 5-nitro-2-(3-phenylpropylamine) benzoic acid (NPPB), the most effective blocker of VacA channels, implying that epithelial permeabilization, like vacuolation, is secondary to the formation of apical anion channels (100). In Caco-2 cells, VacA induces an increased apical anion secretion, and this also is blocked by NPPB (37), implying that it too is due to VacA anion channel formation.

VacA: more than a channel?

VacA has some features in common with A/B-type toxins, which have an active, enzymatic subunit (A) and a binding subunit (B). These features include its putative two-domain structure, the observation that binding is associated with the 58-kDa fragment, and the fact that cytosolic expression of the 37-kDa fragment (admittedly with part of the 58-kDa fragment) induces cell vacuolation. In this A/B toxin model, the active (enzymatic) domain of VacA is hypothesized to be exposed to or released into the cell cytosol, where it modifies an unknown protein involved in the regulation of endosome-endosome fusion. Recently, a 54-kDa VacA-binding protein, called VIP54, has been identified by yeast two-hybrid screening (19). Although VIP54 interacts in vitro with VacA and is associated with the HeLa cell cytoskeleton, so far no evidence of an interaction between VacA and VIP54 in epithelial cells has been provided. Whether or not VacA interacts directly with the cytoskeleton, there is other evidence that VacA-induced cytoskeletal changes may occur. For example, exposure of rat gastric epithelial cells to VacA results in inhibition of actin stress fiber formation and disruption of microtubules (70). Such effects of VacA on cytoskeletal structure are potentially important, not only in dissecting the mechanism of vacuolation, but also in understanding the inhibitory effects of VacA on cell proliferation (71, 84).

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