Helicobacter pylori urease suppresses bactericidal activity of peroxynitrite via carbon dioxide production

Helicobacter pylori can produce a persistent infection in the human stomach, where chronic and active inflammation, including the infiltration of phagocytes such as neutrophils and monocytes, is induced. H. pylori may have a defense system against the antimicrobial actions of phagocytes. We studied the defense mechanism of H. pylori against host-derived peroxynitrite (ONOO^- ), a bactericidal metabolite of nitric oxide, focusing on the role of H. pylori urease, which produces CO₂ and NH₃ from urea and is known to be an essential factor for colonization. The viability of H. pylori decreased in a time-dependent manner with continuous exposure to 1 μM ONOO^-, i.e., 0.2% of the initial bacteria remained after a 5-min treatment without urea. The bactericidal action of ONOO^- against H. pylori was significantly attenuated by the addition of 10 mM urea, the substrate for urease, whereas ONOO^-- induced killing of a urease-deficient mutant of H. pylori or Campylobacter jejuni, another microaerophilic bacterium lacking urease, was not affected by the addition of urea. Such a protective effect of urea was potentiated by supplementation with exogenous urease, and it was almost completely nullified by 10 μM flurofamide, a specific inhibitor of urease. The bactericidal action of ONOO^- was also suppressed by the addition of 20 mM NaHCO₃ but not by the addition of 20 mM NH₃. In addition, the nitration of L-tyrosine of H. pylori after treatment with ONOO^- was significantly reduced by the addition of urea or NaHCO₃, as assessed by high-performance liquid chromatography with electrochemical detection. These results suggest that H. pylori- associated urease functions to produce a potent ONOO^- scavenger, CO₂/HCO₃^-, that defends the bacteria from ONOO^- cytotoxicity. The protective effect of urease may thus facilitate sustained bacterial colonization in the infected gastric mucosa.