Noncatalytic Antioxidant Role for Helicobacter pylori Urease

Abstract

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 ΔureAB 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.

Keywords: gastric pathogen; gastritis; methionine; oxidation; oxidative stress; ulcer.

FIG 1

Urease in cell-free and whole-cell extracts of WT and ureAB mutants. (A) SDS-PAGE (12.5%) analysis of cell-free extract. (B) Immunoblot analysis. Whole-cell extracts were separated via 12.5% SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-UreA antibodies. Strains are indicated above each lane. UreA and UreB are indicated.

FIG 2

Apo-urease confers resistance to HOCl. Cells grown on BA plates with 10 μM NiCl₂ were incubated with PBS or with PBS supplemented with 100 μM HOCl for 1 h at 37°C, diluted, and plated. CFU were counted after 72 h at 37°C. Error bars indicate standard deviations from 4 to 10 independent experiments.

FIG 3

Holo- and apo-urease proteins protect against HOCl. Wild-type and ΔureAB 43504 cells were incubated for 1 h in PBS, PBS with HOCl, or PBS with HOCl that had been previously incubated for 15 min at 37°C with either purified UreAB^WT, UreAB^H138A, UreAB^K219A, or UreE as indicated below each column. Final protein concentration was 0.25 μM, and final HOCl concentration was 200 μM in each reaction. Error bars indicate standard deviations from 3 to 9 independent experiments. ND, no CFU could be detected (detection limit, 10² CFU/ml).

FIG 4

LC-MS/MS analysis of oxidized and Msr-repaired urease. Purified urease was incubated with a 60-fold molar excess of HOCl for 15 min. Excess HOCl was quenched with 15 mM Met and removed via dialysis. After dialysis, oxidized urease was incubated with purified Msr, Trx1, TrxR, NADPH, and DTT for 2 h at 37°C. Urease samples were digested by trypsin, and oxidation levels of methionine residues were quantified by LC-MS/MS. The oxidation level for the untreated sample was below the limit of detection for UreA Met⁵⁸. White bars, untreated; black bars, HOCl treated; gray bars, treated with HOCl plus Msr repair mixture. Error bars indicate standard deviations from 2 independent experiments with 3 replicates each.