Chapter 9Vacuolating Cytotoxin

Atherton JC, Cover TL, Papini E, et al.

Around 1988, Leunk and colleagues discovered that supernatants from broth cultures of Helicobacter pylori induced massive vacuolar degeneration of various cultured epithelial cell lines (53). Since then, the nature of this toxic activity and its role in H. pylori-induced disease have been the subject of intensive study by a number of groups throughout the world. In 1992, the protein mediating the effect was purified and named the vacuolating cytotoxin (10). Determination of the amino-terminal sequence of the protein led, in 1994, to the cloning and sequencing of the toxin gene, which was designated vacA (13, 80, 90, 103). Following the initial characterization of the toxin and its gene, research has focused on VacA structure, the mechanisms underlying VacA's toxic activity, naturally occurring differences among VacA proteins produced by different strains of H. pylori, and the clinical importance of VacA polymorphism.

Interest in VacA has been intense, partly because of its potential as a novel tool for exploring aspects of eukaryotic cell biology, but mainly because of its putative role in the pathogenesis of H. pylori-associated diseases, in particular peptic ulceration and distal gastric adenocarcinoma. The precise role of VacA in these diseases is still under investigation, but VacA may contribute to the capacity of H. pylori to colonize and persist in the human gastric mucosa and may also contribute directly to gastric epithelial damage. Hence, VacA is currently a target for therapeutic intervention and a candidate for inclusion in a vaccine against H. pylori.

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).

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.

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.

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 (5759). 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.

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).

Variation among vacA Alleles in Different H. pylori Strains

vacA evolution

H. pylori exhibits enormous genetic variation, resulting mainly from extensive recombination between H. pylori strains—the most extensive described for any bacterial species (36, 99). For many strain comparisons, this has led to complete linkage equilibrium between genes and also (uniquely among bacteria) between loci within genes (36). Sequence analyses of vacA alleles from different H. pylori strains have shown that there are more pronounced differences between alleles in some regions of the gene than in others (2, 12); for example, a region near the 5′ end of the gene is relatively well conserved between alleles. Within this region, recombination has destroyed virtually all detectable phylogenetic clonal structure and there appears to be linkage equilibrium between most loci (36, 99). The portion of vacA that exhibits maximum diversity is an ~800-bp "mid region," which encodes part of the 58-kDa domain of VacA (Fig. 1) (2, 5). In contrast to the region near the 5′ end of vacA, sequences from the mid region can be phylogenetically divided into two families of alleles, termed m1 and m2 (2, 5). H. pylori strains with type m1 vacA alleles and strains with type m2 alleles are each widespread in all human populations examined, except in the Japanese, among whom type m2 alleles are rare, as further discussed below (3, 44, 118). Strains with evidence of recombination within the vacA mid region between type m1 and m2 alleles appear rare, with m1/m2 recombinant alleles described only in a few Chinese strains of H. pylori (5, 72, 112). Strains with evidence of recombination in the mid region among m1 and among m2 alleles are more frequent, but recombination has not been sufficient to abolish phylogenetic structure even within the m1 and m2 types (5). This residual phylogenetic structure has led to several proposed subdivisions of the main m1 and m2 types (41, 72, 98, 107, 112). Aside from the vacA mid region, various other smaller regions differ markedly between vacA alleles. In particular, the "signal region," encoding part of the amino-terminal signal peptide and the amino terminus of the processed toxin, exhibits striking diversity between strains (Fig. 1) (2). Two main allelic families of signal regions are recognized, termed s1 and s2, and the s1 type can be further subdivided into s1a, s1b, and s1c (2, 109). As a result of extensive recombination among vacA alleles, all combinations of signal and mid regions can theoretically arise (s1a/m1, s1b/m1, s1c/m1, s1a/m2, etc.) and most of these have indeed been described (2, 52, 63, 114).

Analysis of nucleotide substitution rates has provided further interesting insights into vacA evolution. Marked differences in synonymous substitution rates between vacA regions are consistent with extensive recombination and subsequent natural selection of genetic elements within vacA (5). The ratio of nonsynonymous to synonymous substitution rates is a measure of structural constraints on function. This ratio is similar for comparisons among m1 mid regions and among m2 mid regions, but higher for comparisons between m1 and m2 regions. This is consistent with the hypothesis that m1 and m2 regions encode domains with different functions, including perhaps different cell-binding properties, as described below.

vacA polymorphism and toxin activity

Differences between vacA alleles were first investigated in an attempt to understand why culture supernatants from many H. pylori strains did not cause cell vacuolation in vitro, despite all strains having a copy of the toxin gene vacA (2, 12). Nonsense mutations in vacA alleles have been detected, especially in Japan, and these account for absence of toxin activity in some H. pylori strains (44). However, most western isolates of H. pylori express full-length VacA proteins (3), and for such strains, vacA genotype is a major determinant of vacuolating activity. VacA proteins with a type s2 signal sequence undergo amino-terminal signal peptide cleavage at a different site to VacA proteins with a type s1 signal sequence (3). As a consequence, secreted type s2 VacA has a hydrophilic amino terminus, which has been shown to block toxin activity (51). Type s1/m1 VacA proteins are active on a wide variety of epithelial cell lines, whereas at least some tested type s1/m2 VacA proteins, although active on primary gastric epithelial cells and RK-13 cells, are minimally active on HeLa cells (70). This difference in cell type specificity appears attributable, at least in part, to different cell-binding properties mediated by the part of the 58-kDa VacA domain encoded by the vacA mid region (70). Further variations among signal and mid regions beyond the s1/s2 and m1/m2 divisions have not yet been demonstrated to affect VacA toxicity and so are currently of mainly epidemiological and phylogenetic interest.

Association between cagA and vacuolating activity

The close but not absolute association between the presence of the cytotoxin-associated gene, cagA (discussed fully in chapter 31), and vacuolating activity has been enigmatic. In particular, insertional mutagenesis of cagA or deletion of other genes in the cag pathogenicity island (PaI) does not alter VacA expression or activity (1). The discovery of different vacA allelic types has gone some way to explain the link: there is a close genetic association between the presence of cagA (and other genes in the cag PaI) and the presence of type s1 vacA alleles (2). However, given the high level of intraspecies recombination in H. pylori (34, 36, 99) and the separation of vacA and the cag PaI on the H. pylori chromosome, the reason for this genetic association is unclear. It seems likely that there is some as yet unidentified selective advantage to strains that is conferred by either possessing or lacking both type s1 vacA and the cag PaI.

Clinical Importance of Strain Differences in VacA

Association of vacuolating cytotoxin activity with human disease

Numerous studies have shown that H. pylori strains that express vacuolating cytotoxin activity in vitro are more commonly associated with disease than are noncytotoxic strains. Studies from around the world (other than, as discussed below, Japan) have shown an association between vacuolating activity and peptic ulcer disease (2, 27, 35, 82, 102, 114, 122). However, although the association is consistent and significant, it is not absolute. Patients with peptic ulceration frequently have noncytotoxic H. pylori isolates, and patients without ulcers frequently have cytotoxic isolates. Multiple factors complicate the interpretation of these studies, some of which are inherent in all studies examining associations with peptic ulcer disease. For example, patients with peptic ulcer disease may have ulcers in remission at the time of upper gastrointestinal endoscopy and thus a normal endoscopic examination. Similarly, patients with ulcers at endoscopy may be taking nonsteroidal anti-inflammatory drugs, which could be responsible for the ulcers, or alternatively, they may be colonized by both cytotoxic and noncytotoxic strains of H. pylori and have a noncytotoxic strain isolated. A further specific problem with studies assessing vacuolating activity is that these studies have used cell lines only sensitive to type m1 VacA. However, even taking all these points into consideration, it is clear that colonization with cytotoxic strains does not always result in ulcers. Few studies have examined the association between cytotoxic activity and gastric carcinoma or precancerous changes, such as gastric atrophy, but where looked for, weak associations have been found (22, 30).

Relevance of VacA antibodies

Human infection by H. pylori induces a systemic and local antibody response, and antibodies to VacA can be detected by enzyme-linked immunosorbent assays (ELISAs) or immunoblot assays. Commercial kits have been marketed for this purpose. Several studies have assessed the association between the presence of VacA antibodies and disease and usually no association has been found (23, 92, 101, 119). However, some studies have shown weak associations, for example, between VacA expression and peptic ulceration (56) or between the level of antibody response and the level of gastric atrophy (94). The lack of strong associations is not surprising, as the vast majority of strains express a VacA protein, although this may or may not be toxigenic. Recently, purified s1/m1 and s2/m2 VacA proteins have been used in ELISAs, in an effort to distinguish between infection with m1 and m2 strains (79). Developments of this sort may allow more meaningful serological testing for different forms of VacA in the future.

Association of vacA genotypes with disease

The discovery of multiple vacA genotypes and the description of simple and accurate PCR-based methodology for distinguishing between them (2, 3, 108) have stimulated many studies of the association between specific vacA genotypes and disease. The PCR-based methodology means that strain culture is not strictly necessary (38, 87), although many workers have continued to use cultured strains for typing studies. Most such studies from outside Asia have shown that vacA s2 strains are less commonly associated with peptic ulceration or gastric carcinoma than s1 strains (2, 4, 7, 25, 26, 40, 47, 65, 73, 86, 87, 97, 98, 107, 108, 110, 113), although studies from Texas have shown no statistically significant association (34, 118). It is still unclear whether vacA s2 strains are entirely nonulcerogenic. Because of the close genetic association between the vacA s1 signal type and presence of the cag PaI, it is possible that one is merely a marker for the other, although it is equally possible that both are important for disease. A third potential virulence determinant is the blood group antigen-binding adhesin, BabA. The babA gene, like vacA, is polymorphic, and one allelic type, babA2, is disease associated (32). Use of all three markers (vacA s1, cagA, babA2) is a better predictor of pathogenic potential than use of one or two (32), supporting both the concept that all may be clinically important and the idea that the genetic association between them may be due to selective advantage from all being positive, or none.

Most studies have not shown an association between vacA mid region type and disease, although two studies have shown an association of type m1 strains with gastric adenocarcinoma (47, 107). Intriguingly, nearly all strains from Japan are vacA s1/m1 (44, 49, 88, 93, 119), the most toxic type, making it interesting to speculate that toxicity may contribute to the high prevalence of gastric adenocarcinoma in Japan. This homogeneity also means that vacA genotype and presence or absence of toxin activity are not potentially useful markers of more pathogenic strains within Japan.

The mechanisms underlying H. pylori-induced peptic ulceration and gastric adenocarcinoma are unclear. Inflammation, direct toxic activity, or both could be important. The level of inflammation in the gastric mucosa is associated with vacA signal type (s1 > s2) (4, 39). Gastric epithelial damage is also associated with vacA signal type, but one study has shown a further association with vacA mid region type (m1 > m2) (4). The density of H. pylori colonization in vivo may be important in disease pathogenesis, but studies assessing the association of vacA type with H. pylori density have given conflicting results (6, 40, 110). As well as being important in pathogenesis, VacA effects on inflammation and epithelial damage may be important in H. pylori treatment. Antibiotic treatment of H. pylori is more often successful for vacA s1 strains than for vacA s2 strains (9, 110), and one possible explanation for this is that antibiotics are delivered better across inflamed and damaged mucosa.

vacA subtypes as an epidemiological tool

Subtyping of vacA signal and mid regions has given useful information on the population genetics of H. pylori. Of the s1 subtypes of vacA, type s1c is found exclusively in East Asian strains, type s1a is predominant in many northern European countries, and type s1b is predominant in the Iberian Peninsula and South America (106, 107). This latter association suggests genetic relatedness between strains from Spain, Portugal, and South America. Specific m1 and m2 variants have been found in parts of Europe and Asia (41, 72, 98, 107, 112). The main Asian m2 subtype appears specific for this region and, as expected, is commonly associated with the s1c signal type (72, 107, 112). Construction of dendrograms (phylogenetic trees) based on vacA mid region sequences gives further information on relatedness between H. pylori strains from different populations. For example, one analysis shows separate clustering of East Asian, ethnic European, and East Indian strains, implying initial strain dissemination and then relative isolation within these populations (64).

Role of VacA In Vivo

H. pylori produces a toxin that has damaging effects on epithelial cells, and H. pylori colonization is a strong risk factor for the development of peptic ulceration. These facts have led to the hypothesis that VacA directly damages the gastric and duodenal epithelium in vivo and hence causes ulcers. This hypothesis is supported by the association of the s1 type of VacA (which causes vacuolation in vitro) with peptic ulceration and the s2 type (which is nonvacuolating in vitro) with the absence of ulcers. Furthermore, oral administration of VacA to mice causes damage to the gastroduodenal epithelium, including superficial ulceration (33, 103). However, evidence against an epithelial damaging role for VacA in vivo comes from experiments in which piglets or gerbils were infected with wild-type toxigenic strains of H. pylori or VacA null mutants; no differences in epithelial damage were observed (24, 115). This consistent lack of VacA-attributable damage in several infected animal models has led to the suggestion that VacA may offer other, more subtle, advantages to H. pylori in vivo. This suggestion seems logical from an evolutionary viewpoint; it would seem unlikely that a bacterium should produce a toxin primarily to cause a condition such as peptic ulceration from which it does not derive any clear benefit. However, it is feasible that causing ulcers in some individuals is an unfortunate secondary effect of VacA.

One alternative possibility to a directly damaging primary role for VacA is that VacA may play a role in enabling H. pylori to colonize the human stomach. Some evidence supports a role for VacA in gastric colonization; for example, immunization of mice with VacA prevented infection when mice were subsequently experimentally challenged with H. pylori (55). However, other experiments do not support a role for VacA in colonization; wild-type H. pylori strains and VacA null mutants did not differ in their capacity to colonize gnotobiotic piglets (24), and wild-type strains colonized gerbils only slightly more efficiently than did VacA null mutant strains (115).

A further suggestion is that expression of VacA in the human gastric mucosa could potentially offer a survival advantage to H. pylori once colonization is established. Several possible mechanisms have been suggested. VacA-induced epithelial injury or alterations in the integrity of tight junctions might decrease the integrity of the mucosa, and thereby favor H. pylori growth by promoting efflux of nutrients from the mucosa into the mucous layer. Consistent with this hypothesis, VacA induces increases in the permeability of epithelial monolayers in vitro, which results in increased diffusion of critical nutrients such as Ni2+ and Fe3+ (77, 78). The formation of anion-selective channels in the apical membranes of gastric epithelial cells also might facilitate H. pylori growth in the gastric mucosa. For example, the release of HCO3 from cells via VacA channels might help neutralize gastric acidity in the microenvironments where H. pylori is found (100, 104). Finally, by interfering with normal endocytic pathways, VacA may alter the process of antigen presentation, which may be one of the mechanisms by which H. pylori evades host defenses (59). Thus, although considerable attention has been focused on the capacity of VacA to cause tissue injury, the toxin may additionally or alternatively have other, more subtle, functions.

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