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ABSTRACTMaintaining metal homeostasis is crucial for the adaptation of Helicobacter pylorito the gastric environment. Iron, copper, and nickel homeostasis has recently been demonstrated to be required for the establishment of H. pyloriinfection in animal models. Here we demonstrate that the HP0969-0971 gene cluster encoding the Czc-type metal export pump homologs HP0969, HP0970, and the H. pylori-specific protein HP0971 forms part of a novel H. pylorimetal resistance determinant, which is required for gastric colonization and for the modulation of urease activity. Insertional mutagenesis of the HP0971, HP0970, or HP0969 genes in H. pylorireference strain 26695 resulted in increased sensitivity to cadmium, zinc, and nickel (czn), suggesting that the encoded proteins constitute a metal-specific export pump. Accordingly, the genes were designated cznC(HP0971), cznB(HP0970), and cznA(HP0969). The CznC and CznA proteins play a predominant role in nickel homeostasis, since only the cznCand cznAmutants but not the cznBmutant displayed an 8- to 10-fold increase in urease activity. Nickel-specific affinity chromatography demonstrated that recombinant versions of CznC and CznB can bind to nickel and that the purified CznB protein interacted with cadmium and zinc, since both metals competitively inhibited nickel binding. Finally, single cznA, cznB, and cznCmutants did not colonize the stomach in a Mongolian gerbil-based animal model. This demonstrates that the metal export functions of H. pylori cznABCare essential for gastric colonization and underlines the extraordinary importance of metal ion homeostasis for the survival of H. pyloriin the gastric environment.

ABSTRACTThe only known niche of the human pathogen Helicobacter pyloriis the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the H. pylorigenome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in Salmonella entericaserovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the H. pyloriFur homologue in acid resistance. Isogenic furmutants were generated in three H. pyloristrains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the furmutants was severely impaired. No differences were observed between the survival of the furmutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the furmutant at acidic pH. This indicates that the phenotype of the furmutant at low pH was not due to increased iron sensitivity. Transcription of furwas repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of H. pylori; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.