Efficiency of Purine Utilization by Helicobacter pylori: Roles for Adenosine Deaminase and a NupC Homolog

The ability to synthesize and salvage purines is crucial for colonization by a variety of human bacterial pathogens. Helicobacter pylori colonizes the gastric epithelium of humans, yet its specific purine requirements are poorly understood, and the transport mechanisms underlying purine uptake remain unknown. Using a fully defined synthetic growth medium, we determined that H. pylori 26695 possesses a complete salvage pathway that allows for growth on any biological purine nucleobase or nucleoside with the exception of xanthosine. Doubling times in this medium varied between 7 and 14 hours depending on the purine source, with hypoxanthine, inosine and adenosine representing the purines utilized most efficiently for growth. The ability to grow on adenine or adenosine was studied using enzyme assays, revealing deamination of adenosine but not adenine by H. pylori 26695 cell lysates. Using mutant analysis we show that a strain lacking the gene encoding a NupC homolog (HP1180) was growth-retarded in a defined medium supplemented with certain purines. This strain was attenuated for uptake of radiolabeled adenosine, guanosine, and inosine, showing a role for this transporter in uptake of purine nucleosides. Deletion of the GMP biosynthesis gene guaA had no discernible effect on mouse stomach colonization, in contrast to findings in numerous bacterial pathogens. In this study we define a more comprehensive model for purine acquisition and salvage in H. pylori that includes purine uptake by a NupC homolog and catabolism of adenosine via adenosine deaminase

Overview of the current model for purine conversions in <i>H. pylori</i>.

<p>This network allows for salvage of purine nucleobases and nucleosides, as well as inter-conversion between GMP and AMP. <u>Color code:</u> blue; enzymes that have been studied in <i>H. pylori</i> by mutant analysis and/or biochemistry, green; enzymes for which genes have been identified, but whose role has not yet been confirmed, red; putative functional roles whose genetic basis has not yet been identified. <u>Abbreviations:</u> GuaB, IMP dehydrogenase; GuaA, GMP synthetase; GuaC, GMP reductase; PurA, adenylosuccinate synthetase; PurB, adenylosuccinate lyase; Gpt, hypoxanthine-guanine phosphoribosyl-transferase; Apt, adenine phosphoribosyltransferase; DeoD, purine nucleoside phosphorylase; PunB, purine nucleoside phosphorylase; Ade, adenine deaminase; Add, adenosine deaminase; IMP, inosine monophosphate; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; AMP, adenosine monophosphate.</p

Growth of EM207 (Δ<i>nupC</i>) in EMF12 supplemented with a single purine source.

a<p>The initial OD₆₀₀ was standardized to 0.025. Doubling times were calculated using at least four data points taken during exponential growth. Results are the mean ± SD of three independent growth cultures from two independent experiments.</p>*<p>Significantly longer doubling times compared to wild-type (student's t-test P<0.05).</p>b<p>NG  =  No growth observed after 36 hours.</p

Comparison of radiolabeled nucleoside uptake by <i>H. pylori</i> 26695 versus EM207 (Δ<i>nupC</i>).

a<p>Values are the mean ± SEM of four independent growth cultures. Trends were similar among three independent experiments.</p>*<p>Significantly lower uptake compared to wild-type (student's t-test, P<0.01).</p>φ<p>Significantly lower uptake compared to wild-type (student's t-test, P<0.05).</p>Ψ<p>Significant increase in nucleoside uptake for 20 min versus 5 min time point (student's t-test, P<0.05).</p

Sequence comparison between <i>H. pylori</i> NupC homolog (HP1180) and three <i>E. coli</i> CNT paralogs.

<p>Sequences for <i>E. coli</i> YeiJ (GenBank^TM accession number AAA60513.1), <i>E. coli</i> YeiM (GenBank^TM accession number AAA60518.1), HP1180 (GenBank^TM accession number AAD08224.1), and <i>E. coli</i> NupC (GenBank^TM accession number CAA52821.1) were aligned using ClustalW. Membrane-spanning helices were predicted using the TMHMM program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038727#pone.0038727-Krogh1" target="_blank">[46]</a>. Conserved regions typical of CNT transporters are boxed in black <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038727#pone.0038727-Ritzel1" target="_blank">[34]</a>.</p

Growth of <i>H. pylori</i> 26695 in the chemically defined medium EMF12.

<p>Liquid growth medium EMF12 was supplemented with 60 μM hypoxanthine (solid line) or contained no purines (dashed line). <i>H. pylori</i> cells were inoculated at an initial OD₆₀₀ of 0.015 (approx. 1.3×10⁷ cfu/ml). Growth was monitored over time by measuring the absorbance at 600 nm. Results are the mean ± SD of three independent cultures.</p

Growth of <i>gua</i> and <i>pur</i> mutants in EMF12 supplemented with individual purines.

<p><i>H. pylori</i> strains were grown in EMF12 supplemented with one of seven purine sources. After 20 hours, the optical density was measured. Positive growth was defined as a statistically significant increase in OD₆₀₀ relative to the baseline OD₆₀₀ of 0.025 (student's t-test, P<0.05). Results are the mean ± SD of three independent growth cultures.</p

Growth rates and end-point yields of <i>H. pylori</i> grown in EMF12 medium with various purine sources.

a<p>Doubling times were calculated using at least five data points taken during exponential growth. Values are the mean ± SD of three or more independent experiments. Doubling times in guanine, xanthine and guanosine were significantly longer than for hypoxanthine, inosine, or adenosine (student's t-test, P<0.05). Final OD₆₀₀ values were not significantly different from one another.</p>b<p>NG  =  No growth observed after 36 hours.</p

Game dynamics that support snacking, not feasting

\u3cp\u3ePlayer experience research tends to focus on immersive games that draw us into a single play session for hours; however, for casual games played on mobile devices, a pattern of brief daily interaction—called snacking—may be most profitable for companies and most enjoyable for players. To inform the design of snacking games, we conducted a content analysis of game mechanics in successful commercial casual games known to foster this pattern. We identified five single-player game dynamics: Instant Rewards, Novelty, Mission Completion, Waiting, and Blocking. After situating them in theories of motivation, we developed a game in which game mechanics that foster each dynamic can be included individually, and conducted two studies to establish their relative efficacy in fostering the behavioural pattern of snacking, finding significant potential in Novelty and Waiting. Our work informs the design of games in which regular and brief interaction is desired.\u3c/p\u3

Game dynamics that support snacking, not feasting

Player experience research tends to focus on immersive games that draw us into a single play session for hours; however, for casual games played on mobile devices, a pattern of brief daily interaction—called snacking—may be most profitable for companies and most enjoyable for players. To inform the design of snacking games, we conducted a content analysis of game mechanics in successful commercial casual games known to foster this pattern. We identified five single-player game dynamics: Instant Rewards, Novelty, Mission Completion, Waiting, and Blocking. After situating them in theories of motivation, we developed a game in which game mechanics that foster each dynamic can be included individually, and conducted two studies to establish their relative efficacy in fostering the behavioural pattern of snacking, finding significant potential in Novelty and Waiting. Our work informs the design of games in which regular and brief interaction is desired