The Helicobacter pylori toxin VacA causes vacuolar degeneration in mammalian cell lines in vitro and plays a key role in peptic ulcer disease. Two alleles, m1 and m2, of the mid-region of the vacA gene have been described, and the m2 cytotoxin always has been described as inactive in the in vitro HeLa cell assay. However, the m2 allele is associated with peptic ulcer and is prevalent in populations in which peptic ulcer and gastric cancer have high incidence. In this paper, we show that, despite the absence of toxicity on HeLa cells, the m2 cytotoxin is able to induce vacuolization in primary gastric cells and in other cell lines such as RK-13. The absence of Hela cell activity is due to an inability to interact with the cell surface, suggesting a receptor-mediated interaction. This result is consistent with the observation that the m2 allele is found in a population that has a high prevalence of peptic ulcer disease and gastric cancer. VacA is the first bacterial toxin described for which the same active subunit can be delivered by different receptor binding domains.Helicobacter pylori produces a secreted cytotoxin that induces cytoplasmic vacuolation in eukaryotic cells (1-3) and epithelial erosion when administered orally to mice (4). Biological and structural data suggest similarities to the AB family of dichain toxins, which contain an enzymatically active moiety (A) and a receptor binding and translocation moiety (B). VacA is produced as a 140-kDa precursor that is cleaved at the C-terminal domain and released into the extracellular milieu as a 95-kDa mature protein that assembles into large oligomeric structures with hexameric or heptameric radial symmetry (5-6). Each monomer can be cleaved proteolytically at a specific site into two fragments of 37 kDa and 58 kDa that remain associated after cleavage, suggesting that they may represent two distinct cytotoxin subunits (7-8).It has been shown recently that the cytotoxin is able to bind to, and to be internalized by, the target cell (9-10), and a potential receptor has been identified as a membraneassociated protein of 140 kDa (11). Intracellular expression of a transfected vacA gene results in cell vacuolation, indicating activity of the toxin in the cytoplasm (12). The toxicity affects fluid phase endocytosis causing osmotic unbalance and the accumulation of a postendosomal compartment (13)(14). Moreover, VacA interferes with antigen presentation by B cells by impairing processing and maturation of antigens by the antigen-presenting cell (15-16).Only Ϸ50% of clinical isolates of H. pylori produce detectable cytotoxic activity in a HeLa cell vacuolation assay. However, most isolates (Ͼ80%) have a functionally expressed vacA gene. Toxicity has been associated with mosaicism in vacA genes in toxic and nontoxic isolates. Three different signal-peptide sequences (s1a, s1b, and s2) and two variants of the mid-region (m1 and m2) have been described (17-18). Isolates with the s1-m1 forms are toxic, whereas the s2-m2 forms are essentially nontoxic. The m r...

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There are two alleles of the vacuolating cytotoxin gene from Helicobacter pylori, which code for toxins with different cell specificities. By analyzing the phenotypes of natural and artificial chimeras between the two forms of the protein, we have delimited a short stretch of amino acids which determine the cell specificity.

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~ ~Polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells were used to study the pathogenicity of Helicobacterpylori, with an emphasis on the effect of VacA. The adherence of H. pylori to MDCK monolayers resulted in a decrease in trans-epithelial resistance (TER) across the cell monolayer. lsogenic vacA mutants did not lower the TER, demonstrating that the effect is strictly linked to the action of the toxin. A similar effect was observed with all VacA-producing strains, including those producing m2 toxins that are inactive in the vacuolating assay. In contrast to that seen with purified toxin, TER decrease was not enhanced by acid pH, which may indicate that the toxin associated to the bacterial surface is possibly in a monomeric state and therefore does not require a pH-induced conformation to be active. These data raise the possibility that one role of VacA in ulcerogenesis may consist of increasing the paracellular permeability of the gastric epithelium.

In its mature form, the VacA toxin of Helicobacter pylori is a 95-kDa protein which is released from the bacteria as a low-activity complex. This complex can be activated by low-pH treatment that parallels the activity of the toxin on target cells. VacA has been previously shown to insert itself into lipid membranes and to induce anionselective channels in planar lipid bilayers. Binding of VacA to lipid vesicles and its ability to induce calcein release from these vesicles were systematically compared as a function of pH. These two phenomena show a different pH-dependence, suggesting that the association with the lipid membrane may be a two-step mechanism. The secondary and tertiary structure of VacA as a function of pH and the presence of lipid vesicles were investigated by Fourier-transform infrared spectroscopy. The secondary structure of VacA is identical whatever the pH and the presence of a lipid membrane, but the tertiary structure in the presence of a lipid membrane is dependent on pH, as evidenced by H/D exchange.

VacA is a pore-forming cytotoxin produced by Helicobacter pylori in several strain-specific isoforms, which have been classified in two main families, m1 and m2, according to the sequence of a variable "midregion." Both forms are associated with gastric pathologies and can induce vacuolation of cultured cells. The comparison of two representative toxins, m1 17874 and m2 9554, has indicated that the m2 form is less powerful in vacuolation assays and that its effects are more strongly cell type dependent. To rationalize these differences and to investigate structure-function relationships in this toxin, we have compared the properties of the channels formed by these two variants and by a construct derived from 17874 by deleting a loop that connects the two toxin domains, which is shorter in 9554 than in 17874. Although the channels formed by all three proteins are similar, m2 9554 channels have, on average, a lower conductance and are less anion-selective and more voltage-dependent than the m1 pores. Furthermore, the rate of incorporation of 9554 VacA into planar bilayers depends on lipid composition much more strongly than that of 17874. The comparison with the behavior of the loop deletion mutant indicates that this latter property, as well as a portion of the conductance decrease, may be attributed to the reduction in loop length. The differences in pore properties are proposed to account in part for the different cytotoxicity exhibited by the two toxin isoforms. We furthermore present evidence suggesting that the conformation of the membrane-embedded toxin may be influenced by the lipid composition of the membrane itself.

The Helicobacter pylori cytotoxin is proteolytically cleaved at a flexible hydrophilic loop into two subunits. Deletion of the loop sequences had no effect on biological activity of the toxin in the HeLa cell vacuolation assay but favored the organization of the protein into hexameric rather than heptameric structures.