Published online before print March 16, 2006

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(Journal of Leukocyte Biology. 2006;79:1214-1225.)
© 2006 by Society for Leukocyte Biology

Role of urease in megasome formation and Helicobacter pylori survival in macrophages

Justin T. Schwartz^*, and Lee-Ann H. Allen^*,,,1

^* Departments of Medicine and
Microbiology and the
Inflammation Program, University of Iowa and the VA Medical Center, Iowa City

¹Correspondence: Inflammation Program, University of Iowa, 2501 Crosspark Rd., MTF D154, Coralville, IA 52241. E-mail: lee-ann-allen{at}uiowa.edu

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with cat in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH₄Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival.

Key Words: phagocytosis • phagosome maturation • ammonia

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