Helicobacter pylori Relies Primarily on the Purine Salvage Pathway for Purine Nucleotide Biosynthesis

Abstract

Helicobacter pylori is a chronic colonizer of the gastric epithelium and plays a major role in the development of gastritis, peptic ulcer disease, and gastric cancer. In its coevolution with humans, the streamlining of the H. pylori genome has resulted in a significant reduction in metabolic pathways, one being purine nucleotide biosynthesis. Bioinformatic analysis has revealed that H. pylori lacks the enzymatic machinery for de novo production of IMP, the first purine nucleotide formed during GTP and ATP biosynthesis. This suggests that H. pylori must rely heavily on salvage of purines from the environment. In this study, we deleted several genes putatively involved in purine salvage and processing. The growth and survival of these mutants were analyzed in both nutrient-rich and minimal media, and the results confirmed the presence of a robust purine salvage pathway in H. pylori. Of the two phosphoribosyltransferase genes found in the H. pylori genome, only gpt appears to be essential, and an ⌬apt mutant strain was still capable of growth on adenine, suggesting that adenine processing via Apt is not essential. Deletion of the putative nucleoside phosphorylase gene deoD resulted in an inability of H. pylori to grow on purine nucleosides or the purine base adenine. Our results suggest a purine requirement for growth of H. pylori in standard media, indicating that H. pylori possesses the ability to utilize purines and nucleosides from the environment in the absence of a de novo purine nucleotide biosynthesis pathway. Helicobacter pylori is a Gram-negative, microaerophilic helixshaped bacterium that colonizes the gastric mucosa of roughly half of the world's population (19,53). Unlike numerous other pathogenic bacteria capable of existing in diverse environmental niches, H. pylori is only capable of sustained growth within its human host (19). Identified only 27 years ago (45), this bacterium was the first to have two different strains sequenced, allowing for the first genome-wide comparative bioinformatic analysis of a bacterial species (1, 16). The results of this comparative sequencing project (16) as well as subsequent sequencing projects (3) show a relatively small genome with numerous alterations in its metabolic pathways compared with those of other bacterial species. Initial conclusions were that the missing pathway enzymes simply were divergent enough to escape classification by homology screening (16); however, subsequent analysis of the genome has identified 48 potential "dead-end metabolites" (metabolites only consumed or only produced within a metabolic network), indicating missing knowledge about a particular pathway or absence of a fully functional pathway (67). One hypothesis developed to explain these abnormalities was that, having evolved alongside humans for so long, the metabolism of H. pylori was streamlined to coexist in the human niche. Apparent holes in the metabolic pathways of H. pylori suggest the loss of genes no longer required for growth in its relatively stable environment of the huma...

Helicobacter pylori Salvages Purines from Extracellular Host Cell DNA Utilizing the Outer Membrane-Associated Nuclease NucT

“…Bacteria used in all broth culture growth experiments were first grown on sheep blood agar plates (Remel) for 18 h prior to inoculation. All broth cultures were grown in Dulbecco's modified Eagle medium (DMEM) (Gibco) or a derivation of Reynolds and Penn's (RnP) defined media (the composition is provided in Table S1 in the supplemental material) (15,17). RnP medium was made either purine free or containing various concentrations of salmon sperm DNA (Stratagene and Sigma).…”

“…Adenine is the most prominent of the purines in human blood (22), although previous studies examining the effects of purine biosynthesis in H. pylori mutants indicate that, in fetal bovine serum (FBS), the predominant purine available is hypoxanthine, with xanthine being the next most abundant (17). H. pylori mutants capable of growth on only guanine or adenine were incapable of growth in purine-free media supplemented with up to 30% FBS (17), indicating that serum alone does not possess adenine levels that are high enough for the growth requirements of H. pylori.…”

“…Adenine is the most prominent of the purines in human blood (22), although previous studies examining the effects of purine biosynthesis in H. pylori mutants indicate that, in fetal bovine serum (FBS), the predominant purine available is hypoxanthine, with xanthine being the next most abundant (17). H. pylori mutants capable of growth on only guanine or adenine were incapable of growth in purine-free media supplemented with up to 30% FBS (17), indicating that serum alone does not possess adenine levels that are high enough for the growth requirements of H. pylori. Another group has found that an H. pylori strain deleted for guaA, encoding GMP synthase (⌬guaA), is capable of colonizing mice and is indistinguishable from wild-type bacteria in this regard (20), indicating that the levels of either GMP, guanine, or guanosine in the gastric mucosa are sufficient for growth.…”

“…A subsequent study, however, reported that growth could be achieved in the absence of purines if the medium was supplemented with 0.1% catalase in strains NCTC 11639 and N6 and two low-passage-number clinical isolates (16). It was more recently shown that purines are required for growth in Reynolds and Penn's defined medium for strain G27 (17), and that catalase was incapable of restoring growth in purine-free media. Similar studies utilizing the tissue culture medium Ham's F12 and subsequent derivations have also shown that purines are essential for the growth of H. pylori strain 26695 (18,19), and this has been recently reconfirmed by researchers utilizing a series of mutants for purine nucleotide metabolism (20,21).…”

Disruption of de novo ATP biosynthesis abolishes virulence in Cryptococcus neoformans

“…Helicobacter pylori (112) . While this would seem to run contrary to the ubiquity of the pathway in other organisms, the intracellular location of these parasites means that they can exploit their host's purine production, a luxury C. neoformans cannot enjoy due to its purine-poor niche in the central nervous system (32) .…”