Abstract
The production of high levels of ammonia allows the human gastric pathogen Helicobacter pylori to survive the acidic conditions in the human stomach. H. pylori produces ammonia through urease-mediated degradation of urea, but it is also able to convert a range of amide substrates into ammonia via its AmiE amidase and AmiF formamidase enzymes. Here data are provided that demonstrate that the iron-responsive regulatory protein Fur directly and indirectly regulates the activity of the two H. pylori amidases. In contrast to other amidase-positive bacteria, amidase and formamidase enzyme activities were not induced by medium supplementation with their respective substrates, acrylamide and formamide. AmiE protein expression and amidase enzyme activity were iron-repressed in H. pylori 26695 but constitutive in the isogenic fur mutant. This regulation was mediated at the transcriptional level via the binding of Fur to the amiE promoter region. In contrast, formamidase enzyme activity was not iron-repressed but was significantly higher in the fur mutant. This effect was not mediated at the transcriptional level, and Fur did not bind to the amiF promoter region. These roles of Fur in regulation of the H. pylori amidases suggest that the H. pylori Fur regulator may have acquired extra functions to compensate for the absence of other regulatory systems.
Highlights
The human pathogen Helicobacter pylori colonizes the mucus layer overlaying the gastric epithelium, thereby causing persistent gastritis, which can develop into peptic ulcer disease and gastric carcinomas [1]
Orthologs of the corresponding amidase regulatory proteins are absent in H. pylori, the inspection of the H. pylori amiE and amiF promoters indicated the presence of sequences resembling Furboxes, suggesting iron-responsive regulation of these genes [22, 32]
Acid resistance of H. pylori has long been considered to be solely based on unregulated production of large amounts of urease, but recent studies have shown that acid resistance of H. pylori is based on multifactorial, interactive, and probably well regulated processes (3, 25, 34 –36)
Summary
AmiE, aliphatic amidase; AmiF, formamidase; Fur, ferric uptake regulator; BBN, Brucella Broth supplemented with 3% newborn calf serum; BSA, bovine serum albumin; DIG, digoxigenin. Because Fur has been implicated in acid resistance of H. pylori [24] as well as in regulation of urease expression [25], we hypothesized that Fur may regulate the expression of alternative ammonia-producing enzymes. We report that Fur regulates transcription, expression, and activity of the AmiE amidase and indirectly affects enzyme activity of the AmiF formamidase. The regulation of ammonia production via the iron-regulatory protein Fur may be an example of how H. pylori may compensate for its relatively small regulatory capacity
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