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Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity.

ABSTRACT: Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein. NixA, a high-affinity nickel transport protein, allows synthesis of catalytically active urease when coexpressed with H. pylori urease in an Escherichia coli host. To determine whether NixA is essential for the production of active urease in H. pylori, nixA was insertionally inactivated with a kanamycin resistance cassette (aphA) and this construct was electroporated into H. pylori ATCC 43504; allelic exchange mutants were selected on kanamycin-containing medium. The nixA mutation, confirmed by PCR, reduced urease activity by 42% (140 +/- 70 micromol of NH3/min/mg of protein in the mutant versus 240 +/- 100 micromol of NH3/min/mg of protein in the parent (P = 0.037). Rates of nickel transport were dramatically reduced (P = 0.0002) in the nixA mutant (9.3 +/- 3.7 pmol of Ni2+/min/10(8) bacteria) of H. pylori as compared with the parent strain (30.2 +/- 8.1 pmol of Ni2+/min/10(8) bacteria). We conclude that NixA is an important mediator of nickel transport in H. pylori. That residual nickel transport and urease activity remain in the nixA mutant, however, provides evidence for the presence of a redundant transport system in this species.

SUBMITTER: Bauerfeind P

PROVIDER: S-EPMC174160 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity.

Bauerfeind P P Garner R M RM Mobley L T LT

Infection and immunity 19960701 7

Helicobacter pylori, an etiologic agent of gastritis and peptic ulceration in humans, synthesizes urease, a nickel metalloenzyme, as its most abundant protein. NixA, a high-affinity nickel transport protein, allows synthesis of catalytically active urease when coexpressed with H. pylori urease in an Escherichia coli host. To determine whether NixA is essential for the production of active urease in H. pylori, nixA was insertionally inactivated with a kanamycin resistance cassette (aphA) and this ...[more]

PMID: 8698529

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Project description:UnlabelledA novel mechanism aimed at controlling urease expression in Helicobacter pylori in the presence of ample nickel is described. Higher urease activities were observed in an hp0868 mutant (than in the wild type) in cells supplemented with nickel, suggesting that the HP0868 protein (herein named Mua for modulator of urease activity) represses urease activity when nickel concentrations are ample. The increase in urease activity in the Δmua mutant was linked to an increase in urease transcription and synthesis, as shown by quantitative real-time PCR, SDS-PAGE, and immunoblotting against UreAB. Increased urease synthesis was also detected in a Δmua ΔnikR double mutant strain. The Δmua mutant was more sensitive to nickel toxicity but more resistant to acid challenge than was the wild-type strain. Pure Mua protein binds 2 moles of Ni(2+) per mole of dimer. Electrophoretic mobility shift assays did not reveal any binding of Mua to the ureA promoter or other selected promoters (nikR, arsRS, 5' ureB-sRNAp). Previous yeast two-hybrid studies indicated that Mua and RpoD may interact; however, only a weak interaction was detected via cross-linking with pure components and this could not be verified by another approach. There was no significant difference in the intracellular nickel level between wild-type and mua mutant cells. Taken together, our results suggest the HP0868 gene product represses urease transcription when nickel levels are high through an as-yet-uncharacterized mechanism, thus counterbalancing the well-described NikR-mediated activation.ImportanceUrease is a nickel-containing enzyme that buffers both the cytoplasm and the periplasm of Helicobacter pylori by converting urea into ammonia and carbon dioxide. The enzyme is the most abundant protein in H. pylori, accounting for an estimated 10% of the total protein content of the cell, and it is essential for early colonization and virulence. Numerous studies have focused on the transcription of the structural ureAB genes and its control by the regulatory proteins NikR and ArsR. Here we propose that urease transcription is under the control of another Ni-binding protein besides NikR, the Mua (HP0868) protein. Our results suggest that the Mua protein represses urease transcription when nickel levels are high. This mechanism would counterbalance the NikR-mediated activation of urease and ensure that, in the presence of a high nickel concentration, urease activation is limited and does not lead to massive production of detrimental ammonia.

Project description:The well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protect Helicobacter pylori against the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ?ureAB deletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protect H. pylori against oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purified H. pylori urease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCE Chronic Helicobacter pylori infection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid, H. pylori must survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge of H. pylori survival mechanisms.

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Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity.

Publications

Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity.

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