Abstract
The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche.
Highlights
First the UreA/urease B subunit (UreB) structure [15] was analysed in silico to identify surface regions that might tolerate the insertion of two epitope tags (Figure 1a)
Mutant H. pylori with in frame insertions of DNA encoding epitope tag sequences at eight specific sites in chromosomal ureA and ureB genes were generated by a PCR and transformation method
The sites chosen were those corresponding to the N and C-termini of UreA and UreB, respectively and six additional regions in which structural considerations suggested that modest sequence changes would not necessarily inactivate urease’s enzymatic activity
Summary
H. pylori chronically infects the gastric mucosa of billions of people worldwide, causes peptic ulcer disease in 10% or more of them, and is implicated as an early critical risk factor for gastric cancer, one of the most frequently lethal malignancies in human populations [1]. One of the first characterised factors essential for colonisation by H. pylori was urease, an abundant enzyme that decreases the acidity of H. pylori’s immediate environment by generating ammonia and carbonate from the urea we secrete as metabolic waste [2,3]. Such local control of gastric acidity is considered essential, urease-negative H. pylori strains were unable to colonise piglets whose acid secretion had been suppressed, suggesting an additional role for urease [4]. We tested the role of the urease surface in H. pylori/host interactions, and found that surface regions of this enzyme in which changes that did not affect enzymatic activity impaired bacterial persistence in a murine experimental infection model
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