Microbially catalyzed conjugation of GABA and tyramine to bile acids

Abstract

Bile acids (BAs) are cholesterol-derived molecules that aid in digestion and nutrient absorption, regulate host metabolic processes, and influence physiology of the gut microbiota. Both the host and its microbiome contribute to enzymatic modifications that shape the chemical diversity of BAs in the gut. Several bacterial species have been reported to conjugate standard amino acids to BAs, but it was not known if bacteria conjugate BAs to other amine classes. Here, we show that Bacteroides fragilis strain P207, isolated from a bacterial bloom in the J-pouch of a patient with ulcerative colitis pouchitis, conjugates standard amino acids and the neuroactive amines γ-aminobutyric acid (GABA) and tyramine to deoxycholic acid. We extended this analysis to other human gut isolates and identified species that are competent to conjugate GABA and tyramine to primary and secondary BAs, and further identified diverse BA-GABA and BA-tyramine amides in human stool. A longitudinal metabolomic analysis of J-pouch contents of the patient from whom B. fragilis P207 was isolated revealed highly reduced levels of secondary bile acids and a shifting BA amide profile before, during, and after onset of pouchitis, including temporal changes in several BA-GABA amides. Treatment of pouchitis with ciprofloxacin was associated with a marked reduction of nearly all BA amides in the J-pouch. Our study expands the known repertoire of conjugated bile acids produced by bacteria to include BA conjugates to GABA and tyramine and demonstrates that these molecules are present in the human gut. IMPORTANCE BAs are modified in multiple ways by host enzymes and the microbiota to produce a chemically diverse set of molecules that assist in the digestive process and impact many physiological functions. This study reports the discovery of bacterial species that conjugate the neuroactive amines, GABA and tyramine, to primary and secondary BAs. We further present evidence that BA-GABA and BA-tyramine conjugates are present in the human gut, and document a shifting BA-GABA profile in a human pouchitis patient before, during, and after inflammation and antibiotic treatment. GABA and tyramine are common metabolic products of the gut microbiota and potent neuroactive molecules. GABA- and tyramine-conjugated BAs may influence receptor-mediated regulatory mechanisms of humans and their gut microbes, and absorption of these molecules and their entry into enterohepatic circulation may impact host physiology at distal tissue sites. This study defines new conjugated bile acids in the human gut.

Keywords: Bacteroides; GABA; bifidobacteria; bile acid; colitis; gut microbiome; mass spectrometry; metabolomics; neurotransmitter.

Fig 1

Identification of bile acid conjugates produced by pouchitis patient isolate, B. fragilis P207 by UHPLC-MS² (A) Molecular family containing nodes for deoxycholic acid (DCA)-amine conjugates. Colored nodes represent features only observed in B. fragilis P207 cultures with 0.01% (wt/vol) DCA present. The gray-shaded rectangular node is the D₄-DCA-glycine (Gly) internal standard. (B) Structures of the five DCA-amine conjugates with color key for panels A–C. (C) Stacked selected ion chromatograms for the five DCA-amino acid conjugates detected in BHIS media (BHIS), B. fragilis strain P207 culture extract (BHIS + B. fragilis 207), BHIS media with 0.01% DCA (BHIS + DCA), and B. fragilis strain P207 culture extract with DCA (BHIS + B. fragilis P207 + DCA).

Fig 2

Amine feeding experiments to test bile acid conjugation by B. fragilis strain P207. Mass spectrometry analysis was performed to assess production of bile acid conjugates with five different amines: glycine (A), alanine (B), phenylalanine (C), tyramine (D), and GABA (E). B. fragilis P207 was fed isotopically labeled versions of these amines. Observed mass to charge (m/z) shifts, displayed in each panel, are consistent with the expected molecular weights for the respective bile acid-heavy amino acid/amine conjugates presented in Fig. 1.

Fig 3

Extracted ion chromatograms (EICs) of deoxycholate conjugates produced by B. fragilis P207 in vitro compared to synthetic standards. (A) EIC of chemically synthesized deoxycholic acid (DCA)-GABA (m/z 478.3527) (top, retention time = 4.84 min) and the EIC of DCA-GABA identified in B. fragilis P207 in vitro cultures (bottom, retention time = 4.85 min). (B) EIC of chemically synthesized DCA-tyramine (m/z 512.3734) (top, retention time = 6.80 min) and the EIC of DCA-tyramine identified in B. fragilis P207 in vitro cultures (bottom, retention time = 6.81 min). After identifying bile acid conjugates as key metabolites (Fig. 1), we refined the chromatography method to effectively separate the complete array of possible conjugate isomers, considering both DCA and cholic acid isomers as additional potential substrates. This accounts for the differences in retention times from Fig. 1.

Fig 4

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements of amino acid/amine conjugation to the bile acids cholic acid (CA) and deoxycholic acid (DCA), and evidence for deconjugation/re-conjugation of glycodeoxycholic acid (GDCA) by B. fragilis P207. Bar graphs represent the mean area under the curve (AUC) of LC-MS/MS chromatographic peaks corresponding to unconjugated or conjugated bile acids. (A) AUC of bile acid conjugate peaks (labeled below each bar) when B. fragilis P207 was cultivated in BHIS broth the presence of 0.01% (wt/vol) CA. (B) AUC of bile acid conjugate peaks when B. fragilis P207 was cultivated in BHIS broth the presence of CA and DCA [0.01% (wt/vol) total]. (C) AUC of bile acid and bile acid conjugate peaks from B. fragilis P207 culture extract cultivated in BHIS broth the presence of 0.01% (wt/vol) GDCA. Cholate and deoxycholate are known contaminants of the GDCA reagent. Data represent the mean ± SD of two independent cultures. The absence of a bar indicates that a peak corresponding to that chemical species was not detected. (D) Cartoon representing the bile acid conjugate production profile of the human gut isolate, B. fragilis P207.

Fig 5

GC-MS-based detection of tyramine, glycine, alanine, phenylalanine, and GABA in B. fragilis P207 cultures. Cultures were grown in BHIS medium, both in the absence and presence of bile acids, DCA, and GDCA (0.01% wt/vol). The displayed values, derived from the area under the curve (AUC) of detected peaks, represent the relative concentrations of these amine compounds across the different cultures. Each bar represents the mean of three independent experiments, with error bars indicating the standard deviations. Differences between conditions were assessed for statistical significance using analysis of variance with Bonferroni correction. ***A threshold of P < 0.001 was considered to indicate statistical significance.

Fig 6

Molecular network illustrating the diversity and occurrence of bile acid-amine conjugates related to the validated DCA-Phe, DCA-Ala, DCA-Gly, DCA-tyramine, DCA-GABA, CDCA-GABA, CA-GABA, and CA-tyramine products. Each node represents a high-resolution m/z value at a specific retention time. Node quadrant color indicates the sample type that the metabolite was detected in—gray color indicates that the metabolite was not detected in the sample type specified by that quadrant. Edges connect nodes that are related above a score threshold of 25 in Compound Discoverer software suite (Thermo Scientific), with darker edges signifying greater relatedness. Bolded node labels indicate metabolites validated by isotope labeling (GDCA, DCA-Ala, and DCA-Phe) and/or comparison to true synthetic standards (DCA-GABA, DCA-tyramine, CDCA-GABA, CA-GABA, and CA-tyramine). Labels with an asterisk (*) indicate that the node represents a putative bile acid-amine isomer/epimer of the listed metabolite. These isomer assignments are based on comparison of m/z of the intact ion and MS² fragmentation spectra and fragment m/z values to the spectra of the validated bile acid conjugates. All other metabolite nodes were confirmed by comparison to authenticated standards or labeled internal standards. D4 indicates deuterated standards. CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; GCA, glycocholic acid; HCA, hyocholic acid; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; MCA, muricholic acid; TDCA, taurodeoxycholic acid; UCA, ursocholic acid; UDCA, ursodeoxycholic acid.

Fig 7

Extracted ion chromatograms (EICs) of synthetic standards of cholic acid (CA) or chenodeoxycholic acid (CDCA) conjugated to GABA or tyramine compared to stool sample extracts. (A) EIC of chemically synthesized CA-GABA (m/z 494.3476) (top, retention time = 3.20 min) and the corresponding EIC from patient 207 stool at 525 days post functionalization of the ileal pouch (bottom). Peak matching synthetic CA-GABA is marked with a red star. (B) EIC of chemically synthesized CDCA-GABA (m/z 478.3527) (top, retention time = 4.51 min) and the corresponding EIC from patient 207 at 525 days post functionalization of the ileal pouch (bottom). Peak matching CDCA-GABA is marked with a red star. (C) EIC of chemically synthesized CA-tyramine (m/z 528.3864) (top, retention time = 4.31 min) and the corresponding EIC from healthy patient donor (DFI11) stool (bottom). Peak matching synthetic CA-tyramine is marked with a red star. (D) Heatmap illustrating the levels of unconjugated and conjugated bile acids in pouchitis patient 207 (measured by area under the curve) from days 124 to 719 after J-pouch functionalization. DCA (3α,12α-dihydroxy-5β-cholan-24-oic acid) was absent at all time points in this patient, and CDCA was abundant, so we expect the labeled DCA (isomer) amide conjugates have a CDCA core, though the isomeric/epimeric form is not defined in most cases. Likewise, we expect that many of the abundant CA (isomer) conjugates have a cholic acid core but the particular isomer/epimer of these products has not been defined.

Fig 8

UHPLC-MS² measurements of amino acid/amine conjugation to the bile acids cholic acid (CA) and deoxycholic acid (DCA) by M. gnavus, B. longum, B. ovatus, and L. scindens strains isolated from healthy human patients. The bar graphs in this figure represent the mean area under the curve (AUC) of LC-MS/MS peaks corresponding to unconjugated or conjugated CA and DCA (n = 2, errors bars are standard deviations). Normalized AUC of bile acid conjugate peaks (colored according to key) when strains were cultivated in BHIS broth in the presence of 0.01% (wt/vol) DCA or CA. The absence of a bar indicates that a peak corresponding to that chemical species was not detected.