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Immunization with the immunodominant Helicobacter suis urease subunit B induces partial protection against H. suis infection in a mouse model.

ABSTRACT: Helicobacter (H.) suis is a porcine and human gastric pathogen. Previous studies in mice showed that an H. suis infection does not result in protective immunity, whereas immunization with H. suis whole-cell lysate (lysate) protects against a subsequent experimental infection. Therefore, two-dimensional gel electrophoresis of H. suis proteins was performed followed by immunoblotting with pooled sera from H. suis- infected mice or mice immunized with lysate. Weak reactivity against H. suis proteins was observed in post-infection sera. Sera from lysate-immunized mice, however, showed immunoreactivity against a total of 19 protein spots which were identified using LC-MS/MS. The H. suis urease subunit B (UreB) showed most pronounced reactivity against sera from lysate-immunized mice and was not detected with sera from infected mice. None of the pooled sera detected H. suis neutrophil-activating protein A (NapA). The protective efficacy of intranasal vaccination of BALB/c mice with H. suis UreB and NapA, both recombinantly expressed in Escherichia coli (rUreB and rNapA, respectively), was compared with that of H. suis lysate. All vaccines contained choleratoxin as adjuvant. Immunization of mice with rUreB and lysate induced a significant reduction of H. suis colonization compared to non-vaccinated H. suis-infected controls, whereas rNapA had no significant protective effect. Probably, a combination of local Th1 and Th17 responses, complemented by antibody responses play a role in the protective immunity against H. suis infections.

SUBMITTER: Vermoote M

PROVIDER: S-EPMC3542004 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Immunization with the immunodominant Helicobacter suis urease subunit B induces partial protection against H. suis infection in a mouse model.

Vermoote Miet M Van Steendam Katleen K Flahou Bram B Smet Annemieke A Pasmans Frank F Glibert Pieter P Ducatelle Richard R Deforce Dieter D Haesebrouck Freddy F

Veterinary research 20121026

Helicobacter (H.) suis is a porcine and human gastric pathogen. Previous studies in mice showed that an H. suis infection does not result in protective immunity, whereas immunization with H. suis whole-cell lysate (lysate) protects against a subsequent experimental infection. Therefore, two-dimensional gel electrophoresis of H. suis proteins was performed followed by immunoblotting with pooled sera from H. suis- infected mice or mice immunized with lysate. Weak reactivity against H. suis protein ...[more]

PMID: 23101660

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Project description:BackgroundThe sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined.ResultsUrease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35 degrees C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 +/- 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 +/- 0.31 mM and 26.12 +/- 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 +/- 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species.ConclusionWe have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.

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.

Dataset Information

Immunization with the immunodominant Helicobacter suis urease subunit B induces partial protection against H. suis infection in a mouse model.

Publications

Immunization with the immunodominant Helicobacter suis urease subunit B induces partial protection against H. suis infection in a mouse model.

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