A New Helicobacter pylori Vacuolating Cytotoxin Determinant, the Intermediate Region, Is Associated With Gastric Cancer

Abstract

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. 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. Two i-region types were identified, i1 and i2, and both were common among 42 Western clinical isolates. Interestingly, only naturally occurring s1/m2 strains varied in i-type; s1/m1 and s2/m2 strains were exclusively i1 and i2, respectively. Vacuolation assays showed that i-type determined vacuolating activity among these s1/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. 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.