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. 2023 Jun 14;31(6):1038-1053.e10.
doi: 10.1016/j.chom.2023.05.011. Epub 2023 Jun 5.

Gut bacterial metabolism contributes to host global purine homeostasis

Kazuyuki Kasahara  1 , Robert L Kerby  2 , Qijun Zhang  2 , Meenakshi Pradhan  3 , Margarete Mehrabian  4 , Aldons J Lusis  4 , Göran Bergström  5 , Fredrik Bäckhed  6 , Federico E Rey  7
Affiliations

Gut bacterial metabolism contributes to host global purine homeostasis

Kazuyuki Kasahara et al. Cell Host Microbe. .

Abstract

The microbes and microbial pathways that influence host inflammatory disease progression remain largely undefined. Here, we show that variation in atherosclerosis burden is partially driven by gut microbiota and is associated with circulating levels of uric acid (UA) in mice and humans. We identify gut bacterial taxa spanning multiple phyla, including Bacillota, Fusobacteriota, and Pseudomonadota, that use multiple purines, including UA as carbon and energy sources anaerobically. We identify a gene cluster that encodes key steps of anaerobic purine degradation and that is widely distributed among gut-dwelling bacteria. Furthermore, we show that colonization of gnotobiotic mice with purine-degrading bacteria modulates levels of UA and other purines in the gut and systemically. Thus, gut microbes are important drivers of host global purine homeostasis and serum UA levels, and gut bacterial catabolism of purines may represent a mechanism by which gut bacteria influence health.

Keywords: atherosclerosis; gut microbiome; purines; uric acid.

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Conflict of interest statement

Declaration of interests F.B. is co-founder and shareholder of Roxbiosens Inc and Implexion Pharma AB, receives research funding from Biogaia AB, and is a member of the scientific advisory board of Bactolife A/S.

Figures

Figure 1.
Figure 1.. Plasma levels of purines are associated with atherosclerosis burden in transplanted gnotobiotic ApoE KO mice.
A) Experimental design. B-G) Representative sections and quantitative analysis of Oil Red O staining (B, C), MOMA-2 staining (D, E), and Masson’s trichrome staining (F, G) in the aortic sinus (n=11 for AXB10, n=12 for BXD5, n=12 for BTBR group, n=11 for BXA8). The data are expressed as box-and-whisker plots with individual data points, where the boxes indicate the median values and the interquartile ranges and the whiskers represent the minimum and maximum values. Significance was calculated by one-way ANOVA with the Tukey post-tests is indicated as follows: *, p-value <0.05; **, p-value of <0.01; ****, p-value of <0.0001. H) Principal component analysis of gut microbial functions from transplanted mice as determined by metagenomic analysis. I) Plasma metabolites positively or negatively associated with atherosclerotic lesion size, according to Spearman correlation analysis. J) Scatterplots showing associations between purines (relative mass spectrometry scaled intensities) and atherosclerosis lesion size (x104 μm2). GF; germ-free, ApoE; Apolipoprotein E, Chol; cholesterol, MOMA; monocytes and macrophages.
Figure 2.
Figure 2.. Plasma uric acid levels are positively associated with Coronary Artery Calcium (CAC) score in a human cohort.
A) Distribution of uric acid levels in serum from individuals with CAC score=0 and CAC score >0. B) Spearman correlation analysis between serum uric acid levels and CAC score. C) Top 10 taxa associated with serum uric acid levels. Blue and red bars show positive and negative associations, respectively.
Figure 3.
Figure 3.. Gut microbiome modulates purines in cecum and circulation.
A) Heatmap of purines and related metabolites in cecal contents from Conv (n=9) and GF (n=8) mice. B) Values for adenine, xanthine, xanthosine, inosine, hypoxanthine, uric acid, and allantoin in cecal contents measured by LC/MS/MS and C) plasma uric acid levels analyzed by an enzymatic assay are shown using box-and-whisker plots with individual data points, where the boxes indicate the median values and the interquartile ranges and the whiskers represent the minimum and maximum values. Significance was calculated by unpaired two-tailed Student’s t-test and is designated as follows: **, p-value of <0.01; ****, p-value of <0.0001. Conv; Conventionally-raised, GF; germ-free.
Figure 4.
Figure 4.. Gut bacterial isolates use purines as carbon and energy sources.
Anaerobic growth of bacterial strains on plates containing soluble (glucose, allantoin), and insoluble (uric acid and adenine) substrates. As detailed in Methods, plates were inoculated with 4 μl of dense overnight cultures grown in rich medium then incubated for 2 days [no fermentable substrate, glucose, allantoin or uric acid conditions] or 7 days (adenine). Growth is indicated by the appearance of cell patches and a zone of clearing for the overlay plates. Details about strains are specified in the Key Resources Table, and a summary of all tested strains is presented in Supplemental Figure 4. Strains indicated in red were used for colonization of gnotobiotic mice.
Figure 5.
Figure 5.. Purine-degrading bacteria (PDB) modulate abundance of purines in cecum and circulation.
A) Experimental design. Anaerobic uric acid degradation by fecal samples from different groups is indicated using uric acid overlay plates as detailed in Methods. B) Community profiling by sequencing (COPRO-Seq) analysis of fecal samples from gnotobiotic B6 mice colonized with the ‘core’ community (n=4) or the ‘core plus purine-degrading bacteria’ community (n=3). The bar charts show the abundance of each species in each community. C) Heatmap of purines and related metabolites in cecal contents from GF (n=5), ‘core’ (n=3) and ‘core plus PDB’ (n=5) mice analyzed by LC-MS/MS. D) Values for adenine, xanthine, xanthosine, inosine, hypoxanthine, uric acid, and allantoin in cecal contents of the three mouse cohorts analyzed by targeted metabolomics and E) plasma uric acid levels analyzed by enzymatic assay were expressed as box-and-whisker plots with individual data points, where the boxes indicate the median values and the interquartile ranges and the whiskers represent the minimum and maximum values. Significance was calculated by one-way ANOVA test with the Tukey post-tests and is designated as follows: ***, p-value of <0.001; ****, p-value of <0.0001.
Figure 6.
Figure 6.. Identification of a gene cluster necessary for anaerobic bacterial growth on purines.
A) Comparison of transcriptional profiles for Enterocloster bolteae showing differentially-expressed genes (FDR <0.01) and reads per million (RPM)/gene size (kb) for cultures grown on xylose + NH4Cl (“Xylose,” upregulated genes to the left) or uric acid (upregulated genes to the right), highlighting genes encoding two adjacent predicted operons encoding functions likely necessary for uric acid metabolism (blue and red circles). Descriptions of additional upregulated genes including those encoding micronutrient transport, one glycine cleavage system and a probable bifurcating hydrogenase system as well as genes upregulated during growth on xylose are described in Suppl. Fig. 7. B) Diagram of the adjacent upregulated predicted operons, including genes CGC65_RS20560-RS20625, color-matched with the filled circles in panel (A). C) Representative alignments of chromosomal regions from multiple organisms that anaerobically catabolize uric acid. The genetic regions from E. bolteae shown in panel B are compared to those from Clostridioides difficile CD196 (CD196_RS16070 – RS16115), Fusobacterium varium (C4N18_RS01955 – RS01995) and (C4N18_RS03270 – RS03290), Edwardsiella tarda (ETATCC_RS03320 – RS03390) and E. coli MS 200–1 (HMPREF9553_RS03160 - RS03225). Matched genes are color-coded, and the percent similarities of the encoded proteins are indicated. Although selected genes appear to be conserved, their organization differs in different organisms, and in the case of F. varium do not occur in a contiguous chromosomal region. D) Growth of E. coli MS 200–1 wild-type and deletion variants (FER039 [ΔallB::tetA-sacB], FER041 [Δ(ygeW-arcC)::tetA-sacB], and FER063 [ΔygeV::tetA-sacB]) on plates lacking a carbon source, supplemented with glucose or allantoin, or prepared with overlays containing saturating amounts of uric acid, adenine, or hypoxanthine. E) In vivo experimental design. Mice were colonized with the Core community as in Fig. 5 and with either E. coli MS 200–1 wild-type or the deletion variant FER041 [Δ(ygeW-arcC)::tetA-sacB]. Anaerobic uric acid degradation by fecal samples from different groups is indicated using uric acid overlay plates. F) The levels of fecal E. coli in the bacterial communities were assessed by qPCR and (G) plasma uric acid levels measured by HPLC were expressed as box-and-whisker plots with individual data points, where the boxes indicate the median values and the interquartile ranges and the whiskers represent the minimum and maximum values. Significance was calculated by one-way ANOVA test with the Tukey post-tests and is designated as follows: *, p-value of <0.05; ***, p-value of <0.001.
Figure 7.
Figure 7.. Correlation between cecal purines and gut bacterial genes encoding uric acid degradation in transplanted ApoE KO mice.
A) Abundance of genes encoding anaerobic purine degradation in gut metagenomes from gnotobiotic mice transplanted with cecal contents from strains with disparate atherosclerosis phenotypes (see Fig. 1). Differences between groups were evaluated using unpaired two-tailed Welch’s t-test. B) Correlation between abundance of nucleosides and their derivatives and bacterial functions was performed using Spearman correlation.
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