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      Inhibition of Helicobacter pylori urease activity in vivo by the synthetic nickel binding protein Hpn

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          Helicobacter pylori infection is the most common cause of gastroduodenal ulcerations worldwide. Adaptation of H. pylori to the acidic environment is mediated by urease splitting urea into carbon dioxide and ammonia. Whereas neutralization of acid by ammonia is essential for gastric H. pylori colonization, the catalytic activity of urease is mediated by nickel ions. Therefore, nickel uptake and metabolism play key roles in H. pylori infection and urease is considered first line target for drug development and vaccination. Since nickel binding within H. pylori cells is mediated by the Histidine-rich protein designated Hpn, we investigated whether nickel binding by a synthetic Hpn is capable of abrogating urease activity of live H. pylori in liquid cultures. Supplementation of growth media with synthetic Hpn completely inhibited urease acitivity in live cells, indicating that H. pylori nickel uptake is effectively blocked by Hpn. Thus, nickel chelation by Hpn is stronger than nickel uptake of H. pylori offering therapeutic use of Hpn. Although the nickel binding of Hpn was confirmed by binding assays in vitro, its use in anti- H. pylori directed strategy will further need to be adapted to the gastric environment given that protons interfere with nickel binding and Hpn is degraded by pepsin.

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          Most cited references 12

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          The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration.

          Helicobacter pylori produces a 550 kDa, multimeric, nickel-containing urease that catalyses the hydrolysis of urea to yield ammonia and carbonic acid. The ure gene cluster, comprised of seven genes, encodes the two structural subunits UreA (26.5 kDa) and UreB (60.3 kDa), and five accessory proteins: UreI, UreE, UreF, UreG and UreH. Accessory proteins are required for nickel ion insertion into the apoenzyme. The native protein consists of six copies each of UreA and UreB; two nickel ions are coordinated into each UreB active site. Urease is found in the cytosol, but may also localize on the surface (although this may be an artefact) and elicits a strong serum immunoglobulin response. Urease aids in colonization of the host by neutralizing gastric acid and providing ammonia for bacterial protein synthesis. Host defences are avoided by urease by continuing to neutralize acid locally and by shedding urease, which may be bound by immunoglobulin, from the surface of the bacterium. Host tissues can be damaged directly by the urease-mediated generation of ammonia and indirectly by urease-induced stimulation of the inflammatory response, including recruitment of leukocytes and triggering of the oxidative burst in neutrophils.
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            Is Open Access

            Hierarchical regulation of the NikR-mediated nickel response in Helicobacter pylori

            Nickel is an essential metal for Helicobacter pylori, as it is the co-factor of two enzymes crucial for colonization, urease and hydrogenase. Nickel is taken up by specific transporters and its intracellular homeostasis depends on nickel-binding proteins to avoid toxicity. Nickel trafficking is controlled by the Ni(II)-dependent transcriptional regulator NikR. In contrast to other NikR proteins, NikR from H. pylori is a pleiotropic regulator that depending on the target gene acts as an activator or a repressor. We systematically quantified the in vivo Ni2+-NikR response of 11 direct NikR targets that encode functions related to nickel metabolism, four activated and seven repressed genes. Among these, four targets were characterized for the first time (hpn, hpn-like, hydA and hspA) and NikR binding to their promoter regions was demonstrated by electrophoretic mobility shift assays. We found that NikR-dependent repression was generally set up at higher nickel concentrations than activation. Kinetics of the regulation revealed a gradual and temporal NikR-mediated response to nickel where activation of nickel-protection mechanisms takes place before repression of nickel uptake. Our in vivo study demonstrates, for the first time, a chronological hierarchy in the NikR-dependent transcriptional response to nickel that is coherent with the control of nickel homeostasis in H. pylori.
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              Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology.

              Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.

                Author and article information

                European Journal of Microbiology and Immunology
                Akadémiai Kiadó, co-published with Springer Science+Business Media B.V., Formerly Kluwer Academic Publishers B.V.
                1 March 2013
                : 3
                : 1
                : 77-80
                [ 1 ] Institute of Microbiology and Hygiene, Charité - University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany
                [ 2 ] Septomics Research Center, University Hospital Jena, Friedrich-Schiller University, Jena, Germany
                [ 3 ] Institute of Biochemistry, Charité — University Medicine Berlin, Campus Mitte, Berlin, Germany
                [ 4 ] CC5, Institute of Microbiology and Hygiene, Charité — University Medicine Berlin, Campus Benjamin Franklin, Hindenburgdamm 27, D-12203, Berlin, Germany
                Author notes
                [* ] +49-30-8445-2194, +49-30-450-524-902, markus.heimesaat@


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