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Gastroduodenal
PWE-166 Increasing VacA toxin activity alters Helicobacter pylori colonisation density and the nature of the acquired immune response in a mouse model of infection
  1. D P Letley,
  2. J Winter,
  3. A B Greenaway,
  4. K W Cook,
  5. J L Rhead,
  6. J C Atherton,
  7. K Robinson
  1. Nottingham Digestive Diseases Centre Biomedical Research Unit, University of Nottingham, Nottingham, UK

Abstract

Introduction Helicobacter pylori (Hp) infection causes chronic asymptomatic gastritis, and may lead to peptic ulceration and gastric cancer. Hp strains expressing more active forms of the vacuolating cytotoxin VacA are more strongly associated with disease than strains producing less toxigenic VacA. Hp infection stimulates a strong immunosuppressive interleukin-10 (IL-10) response,1 and VacA inhibits T cell activation and interleukin-2 (IL-2) production2 in vitro, but exactly how VacA contributes to disease development remains unclear.

Methods The vacA allelic type of the mouse-colonising Hp strain SS1 was found to be the non-toxigenic s2/i2/m2 form by PCR-typing. To assess whether vacuolating activity affects colonisation, a variant of strain SS1 expressing the more active s1/i1/m2 form of VacA (SS1/s1i1) was constructed. Vacuolating activities were compared in vitro by incubating RK13 epithelial cells with water extracts from each strain for 4 h in the presence of 10 mM ammonium chloride, and counting the number of extensively vacuolated cells in random fields by light microscopy. C57BL/6 mice were infected with either SS1, SS1/s1i1 or given diluent as a placebo. After 3 weeks, gastric colonisation densities were assessed from weighed stomach tissue samples by quantitative culture on selective blood agar plates containing vancomycin, polymyxin B, bacitracin, nalidixic acid and amphotericin B. Immune responses were investigated by culturing spleen cells with and without Hp antigens in vitro and quantifying IL-2 and IL-10 concentrations in culture supernatants by ELISA.

Results As expected, the SS1 wild type strain induced virtually no vacuolation of RK13 cells (2% of cells vacuolated). In contrast, its isogenic variant, SS1/s1i1, expressing the more active form of the toxin, induced vacuolation in 37% of cells (p<0.01). Mutagenesis of SS1 vacA to a more active form had no effect on strain growth and viability in liquid culture media. Mice infected with SS1/s1i1 had approximately 100-fold lower colonisation densities compared to those administered the wild-type strain (p<0.05). Splenocytes from mice infected with SS1/s1i1 secreted 2.8-fold lower IL-2 concentrations and 1.5-fold more IL-10 than cells from wild-type SS1-infected animals in response to stimulation with Hp antigens.

Conclusion Our data show that Hp strains expressing active VacA colonise less densely than less toxigenic strains, and that VacA modulates the immune response in vivo. We speculate that further characterisation of these effects will uncover why strains with active VacA are common in patients developing gastric cancer.

Competing interests None declared.

References 1. Robinson K, Kenefeck R, Pidgeon EL, et al. Helicobacter pylori-induced peptic ulcer disease is associated with inadequate regulatory T cell responses. Gut 2008;57:1375–85.

2. Gebert B, Fischer W, Weiss E, et al. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 2003;301:1099–102.

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