Differential Regulation of Amidase- and Formamidase-mediated Ammonia Production by the Helicobacter pylori Fur Repressor

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.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). H. pylori is able to survive and colonize this hostile acidic niche, aided by the expression of its acid resistance mechanisms (2, 3). One of the major factors contributing to acid resistance of H. pylori is the production of ammonia by its urease enzyme, which is essential for gastric colonization in different animal models (4 -7). However, the role of urease in gastric colonization extends beyond protection against gastric acid, because H. pylori urease mutants are still unable to colonize the gastric mucosa when gastric acid production is abolished with proton pump inhibitors (4).Ammonia is a key component of bacterial nitrogen metabolism, because it is the preferred source of nitrogen for the synthesis of amino acids, pyrimidines, and purines. Ammonia plays a central role in pathogenesis and metabolism of the important human pathogen H. pylori, because it not only serves as nitrogen source (8) but also contributes to epithelial cell damage and apoptosis (9, 10), is involved in chemotactic motility (11), and is required for acid resistance (2, 3). Urea is thought to be the main source of ammonia in the gastric environment, but H. pylori does have alternative pathways for the production of ammonia via amino acid catabolism (12) and via the activity of its two paralogous amidases, AmiE 1 and AmiF (13, 14). Aliphatic amidase (AmiE, EC 3.5.1.4) and formamidase (AmiF, EC 3.5.1.49) catalyze the conversion of amide substrates to the corresponding carboxylic acid and ammonia...

Characterization of the ArsRS Regulon ofHelicobacter pylori, Involved in Acid Adaptation

“…Under ironreplete conditions, transcription of amiE has been shown to be repressed by Fur, which binds to the upstream region of amiE. To our knowledge, amiF transcription is not controlled by Fur (39). However, at low pH the activity of both amidases is conversely affected by the inactivation of Fur or the NikR protein, suggesting a role for both regulators in the acid-responsive regulation of the amidases (6,40).…”

Transcriptional profiling of Helicobacter pylori Fur- and iron-regulated gene expression

“…1b). The amiE gene was previously demonstrated to be iron-and Fur-repressed (van Vliet et al, 2003), and this regulation is apparent at each of the three time-points (Fig. 1b).…”

“…These included genes putatively involved in: i) biosynthesis of cofactors and prosthetic groups, including biotin (bioB), isoprenoid (ispE) and pyridoxal phosphate (pdxA); ii) production of cell envelope and surface structures, such as the murB peptidoglycan synthesis gene and the flaB and fliP flagellar biosynthesis genes (Josenhans et al, 2000); iii) energy metabolism, with both the paralogous amidases amiE and amiF (Skouloubris et al, 2001;van Vliet et al, 2003); iv) protein synthesis, in which the 16S rRNA dimethyltransferase gene ksgA is putatively involved. Finally, expression of the hypothetical protein HP906 was repressed by Fe-Fur.…”

“…Other than regulation of iron metabolism, H. pylori Fur has also been implicated in the regulation of acid resistance (Bijlsma et al, 2002;Bury-Mone et al, 2004;van Vliet et al, 2004), nitrogen metabolism (van Vliet et al, 2001(van Vliet et al, , 2003 and oxidative stress resistance (Barnard et al, 2004;Cooksley et al, 2003;Harris et al, 2002). Fur-mediated regulation is also required for gastric colonization by H. pylori, as demonstrated in a mouse model of infection (Bury-Mone et al, 2004).…”

Requirement of Histidine Kinases HP0165 and HP1364 for Acid Resistance in Helicobacter pylori

“…The diminished acid resistance of HP0165 and HP1364 mutant strains compared to the wildtype strain may be due to changes in the expression of several genes, including those that encode urease, amidase (HP0294), and formamidase (HP1238) ( Table 3). These enzymes, because of their capacity to generate ammonia, are all likely to play a role in acid resistance (7,35,37).…”