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The Helicobacter pylori vacuolating cytotoxin gene, vacA, is naturally polymorphic, the two most diverse regions being the signal region (which can be type s1 or s2) and the mid region (m1 or m2). Previous work has shown which features of vacA make peptic ulcer and gastric cancer-associated type s1/m1 and s1/m2 strains toxic. vacA s2/m2 strains are associated with lower peptic ulcer and gastric cancer risk and are non-toxic. We now define the features of vacA that determine the nontoxicity of these strains. To do this, we deleted parts of vacA and constructed isogenic hybrid strains in which regions of vacA were exchanged between toxigenic and non-toxigenic strains. We showed that a naturally occurring 12-amino acid hydrophilic N-terminal extension found on s2 VacA blocks vacuolating activity as its removal (to make the strain s1-like) confers activity. The mid region of s2/m2 vacA does not cause the non-vacuolating phenotype, but if VacA is unblocked, it confers cell line specificity of vacuolation as in natural s1/m2 strains. Chromosomal replacement of vacA in a nontoxigenic strain with vacA from a toxigenic strain confers full vacuolating activity proving that this activity is entirely controlled by elements within vacA. This work defines why H. pylori strains with different vacA allelic structures have differing toxicity and provides a rational basis for vacA typing schemes.

Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA+ background.

Persistent Helicobacter pylori infection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylori facilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pylori MV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pylori strains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pylori MV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pylori MV in the stimulation of innate pro-and anti-inflammatory responses and in the suppression of T cell immunity.

Helicobacter pylori infections are usually established in early childhood and continuously stimulate immunity, including T-helper 1 (Th1), Th17, and regulatory T-cell (Treg) responses, throughout life. Although known to be the major cause of peptic ulcer disease and gastric cancer, disease occurs in a minority of those who are infected. Recently, there has been much interest in beneficial effects arising from infection with this pathogen. Published data robustly show that the infection is protective against asthma in mouse models. Epidemiological studies show that H. pylori is inversely associated with human allergy and asthma, but there is a paucity of mechanistic data to explain this. Since Th1 and Treg responses are reported to protect against allergic responses, we investigated if there were links between the human systemic Th1 and Treg response to H. pylori and allergen-specific IgE levels. The human cytokine and T-cell responses were examined using peripheral blood mononuclear cells (PBMCs) from 49 infected and 58 uninfected adult patients. Concentrations of total and allergen-specific plasma IgE were determined by ELISA and ImmunoCAP assays. These responses were analyzed according to major virulence factor genotypes of the patients’ colonizing H. pylori strains. An in vitro assay was employed, using PBMCs from infected and uninfected donors, to determine the role of Treg cytokines in the suppression of IgE. Significantly higher frequencies of IL-10-secreting CD4+CD25hi Tregs, but not H. pylori-specific Th1 cells, were present in the peripheral blood of infected patients. Total and allergen-specific IgE concentrations were lower when there was a strong Treg response, and blocking IL-10 in vitro dramatically restored IgE responses. IgE concentrations were also significantly lower when patients were infected with CagA+ strains or those expressing the more active i1 form of VacA. The systemic IL-10+ Treg response is therefore likely to play a role in H. pylori-mediated protection against allergy in humans.

The Helicobacter pylori vacuolating cytotoxin VacA shares homology in its C-terminal domain with many autotransporter proteins, suggesting a similar mechanism of secretion. Like most autotransporters, VacA contains a single pair of cysteine residues located near the C-terminus of the passenger domain. This study aimed to investigate the role of these conserved cysteine residues. This involved changing each cysteine in the VacA passenger domain to serine, quantifying the effect on VacA levels and assessing toxin activity in H. pylori. It was shown that both cysteine residues were required for high VacA levels, although mutation of each cysteine reduced toxin amounts to differing extents, implying that their importance was not simply for intramolecular disulphide bond formation. Although less VacA was observed for the cysteine mutants, vacuolating activity was detected, showing that the cysteines were not required for VacA function. INTRODUCTIONChronic gastric colonization by Helicobacter pylori is the main cause of peptic ulcer disease and a significant risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma (Parsonnet et al., 1994(Parsonnet et al., , 1991Warren & Marshall, 1983). Although more than half the world population is infected with H. pylori, most people remain asymptomatic. Who develops disease depends on strain virulence, host genetic susceptibility and environmental factors. One of the major H. pylori virulence factors which affects disease progression is the vacuolating cytotoxin VacA (Figura et al., 1989;Tee et al., 1995), which induces extensive cytoplasmic vacuolation in epithelial cells (Leunk et al., 1988), permits cytochrome c release from mitochondria leading to apoptosis (Galmiche et al., 2000), causes gastro-duodenal damage in a mouse model (Marchetti et al., 1995) and increases gastric ulcer risk in H. pyloriinfected Mongolian gerbils (Ogura et al., 2000). The vacuolating activity of VacA has been extensively studied and shown to be dependent on the insertion of VacA multimers into cell membranes to form anion-selective pores Tombola et al., 1999). The mechanism of subsequent vacuole formation and the cellular origin of the vacuoles from late endosomes has been characterized (Cover et al., 1993;Molinari et al., 1997; Papini et al., 1993a Papini et al., , b, 1994Papini et al., , 1996Ricci et al., 1997).The vacA gene encodes a preprotoxin of~139 kDa (Cover et al., 1994;Phadnis et al., 1994;Schmitt & Haas, 1994;Telford et al., 1994) which includes an N-terminal signal peptide and an~50 kDa C-terminal domain that remains associated with the cell following secretion (Telford et al., 1994). The secreted toxin monomer is 87-95 kDa (Cover & Blaser, 1992;Telford et al., 1994). Such a genetic structure is characteristic of an autotransporter and, indeed, the C-terminal, cell-associated domain shares homology with other autotransporters (Loveless & Saier, 1997). Autotransporters are secreted proteins, generally assumed to direct their own transport across the ou...

We show here that Helicobacter pylori broth culture supernatants disrupt the actin cytoskeleton of epithelial cell lines, leading to cell rounding and apoptosis through anoikis. We demonstrate that there are marked quantitative differences between strains and that there are different cell line sensitivities. By constructing VacA null isogenic mutants, we show that the effect is not due to the vacuolating cytotoxin.Helicobacter pylori is the main cause of peptic ulceration and the most important risk factor for distal gastric adenocarcinoma and primary gastric lymphoma (22,23,26). Although about 30% of the population in developed countries and more than 60% of the population in developing countries are infected with H. pylori, most individuals do not develop significant disease. Who develops disease depends on host genetic factors, (11) environmental factors (19), and bacterial virulence determinants, such as vacuolating cytotoxin (VacA) activity and possession of the cag pathogenicity island (1, 4, 5). However, the pathogenesis of H. pylori-associated diseases remains uncertain. One much-studied area is H. pylori-induced epithelial cell apoptosis (6,14,20,28,30). Apoptotic cell loss could be a factor in both gastroduodenal ulceration and gastric atrophy; the latter is a known precursor of gastric adenocarcinoma (7). Disruption of the proliferation-apoptosis balance could also be important in affecting the number of surviving cells with DNA replication errors and so in modulating cancer risk. Isogenic mutant studies have shown that the cag pathogenicity island may play a role in inducing epithelial cell apoptosis (16), although cag-negative strains can also induce apoptosis (30). VacA is another strong inducer of apoptosis, as shown by experiments in which bacterial cell-free culture supernatants from wild-type strains and isogenic VacA-negative mutants were compared (14).While studying VacA-induced epithelial cell apoptosis using bacterial culture supernatants, we noticed that among the rounded apoptotic cells were other cells, which, although rounded, did not show features of apoptosis. In this study we characterized this newly described effect of H. pylori on epithelial cells, showed that it is bacterial strain and epithelial cell line dependent, and proved, using an isogenic mutant approach, that it is not VacA dependent.Preparation of H. pylori broth culture supernatants. We used the H. pylori strains described in Table 1 (3, 4, 10, 17). We prepared concentrated broth culture supernatants of H. pylori from 48-h cultures in sulfite-free brucella broth (2) supplemented with 0.2% ␤-cyclodextrin and incubated microaerobically (Campypak-Plus; Becton-Dickenson) with shaking at 37°C. We adjusted the optical density at 550 of uncontaminated broth media to 0.8, removed bacterial cells by centrifugation, concentrated filtered supernatants 20-fold by ultrafiltration with Centriprep-30 filters (Amicon), and filtered (pore size, 0.2 m) the preparations before dividing them into aliquots and freezing them at Ϫ20°C.H. pylo...