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Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

ABSTRACT: "Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular weights of 25,729 and 61,831, respectively. The urease subunit genes from "H. heilmannii" were cloned and expressed in Escherichia coli. Western blot (immunoblot) analysis showed that antiserum directed against the ureA and ureB gene products from H. pylori was cross-reactive with the corresponding polypeptides from "H. heilmannii." Analysis of the derived amino acid sequences of "H. heilmannii" UreA and UreB demonstrated that "H. heilmannii" urease is more highly related to the urease from H. felis (found in the stomachs of cats and dogs) than to the urease from H. pylori. These data are consistent with 16S rDNA sequence analysis and suggest that "H. heilmannii" is phylogenetically most closely related to H. felis.

SUBMITTER: Solnick JV

PROVIDER: S-EPMC186371 | biostudies-other | 1994 May

REPOSITORIES: biostudies-other

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Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

Solnick J V JV O'Rourke J J Lee A A Tompkins L S LS

Infection and immunity 19940501 5

"Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular ...[more]

PMID: 8168924

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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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Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

Publications

Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

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