Helicobacter pylori rocF Is Required for Arginase Activity and Acid Protection In Vitro but Is Not Essential for Colonization of Mice or for Urease Activity

Abstract

Arginase of the Helicobacter pylori urea cycle hydrolyzes l-arginine to l-ornithine and urea.H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved inH. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori genehp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 androcF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was ∼1,000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of l-arginine, in contrast to the WT, and yielded a ∼10,000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylorimouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log10 CFU) were 6.1, 5.5, and 4.1, respectively. Thus,H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.

Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily

“…2A), (c) transforming E. coli with a plasmid containing disrupted rocF abolished arginase activity ( Fig. 2A) [8]. These results demonstrate that rocF is necessary and sufficient for arginase activity.…”

“…Five lines of evidence indicated that the rocF gene encodes arginase, namely (a) disrupting the rocF gene in H. pylori abolishes arginase activity ( Fig. 1) [8], (b) transforming E. coli with a plasmid containing rocF conferred arginase activity ( Fig. 2A), (c) transforming E. coli with a plasmid containing disrupted rocF abolished arginase activity ( Fig.…”

“…In a previous study, NMR spectroscopy was employed to measure arginase activity [8]. A colorimetric assay, described previously [11], which allows higher throughput, was utilized and further developed in this study.…”

“…Previously, the rocF gene of H. pylori was cloned and the gene disrupted in three strains [8]. While the gene was found to be required for arginase activity, the study did not establish whether expression of rocF alone in Escherichia coli was sufficient for arginase activity.…”

“…Previous studies have pointed to the role of H. pylori arginase in acid resistance [8]. H. pylori arginase also inhibits host nitric oxide production, helping to escape the toxic effects of nitric oxide, while outcompeting the host for the limited arginine available [7].…”