A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer

Background & Aims: Helicobacter pylori is the main cause of peptic ulceration and gastric adenocarcinoma. The vacuolating cytotoxin gene, vacA, is a major determinant of virulence. Two naturally polymorphic sites in vacA, the signal region and midregion, are well-characterized determinants of toxicity and markers of pathogenesis. The aim of this study was to characterize a new vacA polymorphic site, the intermediate (i) region. Methods: The vacA i-region was identified and characterized by constructing isogenic vacA exchange mutants and determining their vacuolating activity on HeLa, AGS, and RK13 cell lines. The vacA i-region types of H pylori isolates from patients undergoing routine endoscopy were determined by nucleotide sequencing and allele-specific polymerase chain reaction. Results: Two i-region types were identified, il and i2, and both were common among 42 Western clinical isolates. Interestingly, only naturally occurring sl/m2 strains varied in i-type; sl/ml and s2/m2 strains were exclusively il and i2, respectively. Vacuolation assays showed that i-type determined vacuolating activity among these sl/m2 strains, and exchange mutagenesis confirmed that the i-region itself was directly responsible. Using a simple i-region polymerase chain reaction-based typing system, it was shown for 73 Iranian patients that i1-type strains were strongly associated with gastric adenocarcinoma (P < 10(-3)). Finally, logistic regression analysis showed this association to be independent of, and larger than, associations of vacA s- or m-type or cag status with gastric adenocarcinoma. Conclusions: Together these data show that the vacA i-region is an important determinant of H pylori toxicity and the best independent marker of VacA-associated pathogenicity.