Role of Hpn and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal ions

Background. Helicobacter pylori produces Hpn, a 60-amino acid, histidine-ri ch protein that avidly binds nickel and zinc ions, and NixA, a high-affinit y nickel transporter in the cytoplasmic membrane. We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H. pylori. Materials and Methods. Hpn-negative mutants of four H. pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn()/Hpn-deficient pairs. A metal concentration that inhibited growth by 50% ( IC50) was calculated for Ni2+, Zn2+, Cu2+, and Co2+ by comparing OD600 of c ultures in metal-supplemented and control media. Results. Among all four pairs of isogenic strains, the tolerance for Ni2+ w as reduced significantly (p < .001) in the Hpn mutants; the mean IC50 value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM. In contra st, growth inhibition by Zn2+ was identical within the fours pairs, as was Cu2+ and Co2+ tolerance in one pair tested. We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by te sting a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics. Minimal inhibitory concentrations of ranitid ine bismuth citrate dropped from 9.2 to 2.3 mu g/ml, and those of bismuth c itrate dropped from 7.4 to 3.2 mu g/ml (p < .05 for both comparisons), whil e susceptibility to the antibiotics was unaffected. Disruption of the nixA gene encoding the specific Ni2+ transport protein of H, pylori did not chan ge susceptibility to bismuth. Conclusion. We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.