Characterization of the Vacuolating Cytotoxin in Helicobacter pylori Strains Isolated from Iran

Abstract

Objective: Helicobacter pylori (H. pylori) cytotoxin and its heterogeneity amongst strains hasbeen closely linked to the varying infection-associated clinical outcomes. In order to determinethe decisive role of the vacuolating cytotoxin (vacA) gene mosaicism in its corresponding geneexpression and phenotype, we aimed to characterize vacA alleles of different H. pylori strainsin addition to the resulting protein and its vacuolating activity in epithelial cell culture.Materials and Methods: vacA gene polymorphism was determined for 80 H. pylori strainsisolated from dyspeptic patients, using multiplex gene-specific polymerase chain reaction(PCR). VacA protein was detected by immuno-blotting assay using a polyclonal anti-VacAantibody. In vitro cytotoxicity assay was conducted on HeLa cells in order to evaluate thevacuolating cytotoxin activity.Results: Genotyping revealed the following strain distribution: 26 (32.5%) s1m1, 35(43.8%) s1m2, and 19 (23.8%) s2m2 subtypes. Infection with s1m1 type strain was significantlyassociated with gastric cancer as compared to non-ulcer dyspepsia (p=0.005)and peptic ulcer disease (p=0.008). A 95-kDa immuno-reactive band that represented thevacuolating toxin was demonstrated in SDS-PAGE analysis of concentrated culture filtrate(CCF) of H. pylori strains. H. pylori CCFs induced HeLa cell vacuolation which correlatedwith the strain genotype; s1m1 strains demonstrated higher levels of vacuolation as comparedto s1m2 strains, whereas s2m2 strains showed no detectable cytotoxic activity.Conclusion: The current study confirmed the relatively high cytotoxic activity of s1m1type H. pylori strains which infect the majority of patients suffering from gastric cancer andmay be partly responsible for the pathogenesis of this mortal disease.

Keywords

• Vacuolation
• Cytotoxins
• Genotype
• Virulence
• Helicobacter pylori