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. Author manuscript; available in PMC: 2022 Oct 28.
Published in final edited form as: Nat Microbiol. 2022 Apr 28;7(5):680–694. doi: 10.1038/s41564-022-01103-1

Figure 6: Microbiota mediates PA suppression through microbial β-glucuronidase enzymatic activity and production of unconjugated bilirubin.

Figure 6:

a, Beta-glucuronidase (GUS) activity in humans. High PA PI-IBS had lower fecal GUS activity compared to healthy and low PA PI-IBS (One-Way ANOVA-Kruskal-Wallis, *p=0.02 and *p=0.03 respectively, n=4 low PA, 8 high PA and 8 healthy). b, Concentration of urobilinogen in human feces. Untargeted fecal metabolomics identified higher urobilinogen in low PA compared to high PA feces (n=3 high PA and 4 low PA). c, Liquid chromatography tracing of urobilinogen (C18 column, n=3 high PA and 4 low PA PI-IBS). d, GUS suppresses PA in mice. Mice monocolonized with E. coli overexpressing GUS (GUS+) had significantly lower fecal PA compared to controls (1437 ± 411.7 vs. 3371 ± 481.9 BAEE units/mg protein, One-Way ANOVA-Kruskal-Wallis, p=0.004, n=5). e, Unconjugated bilirubin suppresses PA. Mice given unconjugated bilirubin (gavage, 3 days) had less trypsin-like activity compared to controls (4.8 vs 12.2 nmoles/min/μg protein, Mann-Whitney p=0.03, n=4 mice). f, Fecal metabolomics in experimental mice. Increased D-urobilin, #p=0.001 and bilirubin degradation product, C17H20N2O5 #p=0.03, seen in GUS+ and D-Glucaro-1–4-Lactone treated animals respectively (n=4 mice). g, Schematic for GUS inhibitors in humanized mice. h, D-Glucaro-1–4-Lactone increases fecal PA. Mice given D-Glucaro-1–4-Lactone, a non-specific GUS inhibitor, have significantly higher fecal PA than controls (825.3 ± 655.6 vs. 19.3 ± 8.0 BAEE units/mg protein, Mann-Whitney, n=5, p=0.008). i, UNC10201652, a microbial GUS inhibitor, increases fecal PA (2692 ± 2430 vs. 113.8 ± 230.1 BAEE units/mg of protein, Mann-Whitney, n=5, p=0.008). Data presented mean ± s.d. Boxplots as previously described. Human and mouse metabolite intensities were normalized using total ion current for each sample, log-transformed and compared between groups (Student’s t-test). p-values were adjusted using Benjamini-Hochberg method. Metabolites p-value <0.05 and absolute log2 fold change of > 0.5 (0.0 = no change) are mapped.