Introduction The Gram negative bacterium Helicobacter pylori persistently colonises the stomachs of around half the world's population. It causes chronic asymptomatic gastritis, and leads to peptic ulceration and gastric cancer in a small proportion of cases. One of the key factors influencing the development of more severe pathology and disease is the expression of virulence factors by the colonising strain. The vacuolating cytotoxin, VacA, is a major determinant of H pylori pathogenicity and is highly polymorphic. Strains producing more active forms of VacA (i1 types) are more strongly associated with gastric cancer than strains producing less toxigenic VacA (i2 types).1 The i-region polymorphism is characterised by 10 amino acid substitutions and a serine-asparagine-glutamine (SNQ) tri-peptide insertion in i2. We aimed to further characterise the structure and function of this important region.
Methods The SNQ tri-peptide from i2 was engineered into H pylori strain 60190 (VacA s1/i1/m1) to make strain 60190/SNQ. The vacuolating activities of 60190/SNQ and wild type strains were compared by incubating AGS cells with broth culture supernatants, and counting vacuolated cells in random fields by microscopy. To allow further structure-function characterisation of i-region variants, we modified a recombinant VacA system2 by expressing VacA p33 and p55 subunits in Escherichia coli and purifying both under denaturing conditions by metal affinity chromatography, then refolding by stepwise dialysis. Recombinant VacA was tested for vacuolating activity and previously described effects on Jurkat T-cells.3
Results Introduction of the i2-specific SNQ insertion into i1 VacA in the H pylori 60190 strain resulted in a 1.8-fold decrease in native toxin vacuolating activity on AGS cells lines (pH pylori—they induced extensive vacuolation of gastric epithelial cell lines at concentrations down to 0.2 μg/ml. The recombinant toxin also inhibited proliferation of, and IL-2 production by Jurkat cells. Engineering the SNQ insertion into recombinant i1 VacA resulted in modest reductions in the stability of the toxin.
Conclusion A naturally-occurring three amino acid insertion in the VacA i-region renders the toxin less active in vitro. We speculate that this small insertion is a major contributor to the reduced association with cancer of strains with type i2 VacA. Recombinant VacA toxin is highly active on both epithelial and T cells and can be genetically modified to explore VacA structure-function relationships. We aim to use this system to further characterise the structure and function of i-region variants.
Competing interests None declared.
References 1. Rhead JL, Letley DP, Mohammadi M, et al. A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology 2007;133:926–36.
2. González-Rivera C, Gangwer KA, McClain MS, et al. Reconstitution of Helicobacter pylori VacA toxin from purified components. Biochemistry 2010;49:5743–52.
3. Gebert B, Fischer W, Weiss E, et al. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 2003;301:1099–102.