Dominant Bacterial Phyla from the Human Gut Show Widespread Ability To Transform and Conjugate Bile Acids

Abstract

Gut bacteria influence human physiology by chemically modifying host-synthesized primary bile acids. These modified bile acids, known as secondary bile acids, can act as signaling molecules that modulate host lipid, glucose, and energy metabolism and affect gut microbiota composition via selective antimicrobial properties. However, knowledge regarding the bile acid-transforming capabilities of individual gut microbes remains limited. To help address this knowledge gap, we screened 72 bacterial isolates, spanning seven major phyla commonly found in the human gut, for their ability to chemically modify unconjugated bile acids. We found that 43 isolates, representing 41 species, were capable of in vitro modification of one or more of the three most abundant unconjugated bile acids in humans: cholic acid, chenodeoxycholic acid, and deoxycholic acid. Of these, 32 species have not been previously described as bile acid transformers. The most prevalent bile acid transformations detected were oxidation of 3α-, 7α-, or 12α-hydroxyl groups on the steroid core, a reaction catalyzed by hydroxysteroid dehydrogenases. In addition, we found 7α-dehydroxylation activity to be distributed across various bacterial genera, and we observed several other complex bile acid transformations. Finally, our screen revealed widespread bacterial conjugation of primary and secondary bile acids to glycine, a process that was thought to only occur in the liver, and to 15 other amino acids, resulting in the discovery of 44 novel microbially conjugated bile acids. IMPORTANCE Our current knowledge regarding microbial bile acid transformations comes primarily from biochemical studies on a relatively small number of species or from bioinformatic predictions that rely on homology to known bile acid-transforming enzyme sequences. Therefore, much remains to be learned regarding the variety of bile acid transformations and their representation across gut microbial species. By carrying out a systematic investigation of bacterial species commonly found in the human intestinal tract, this study helps better define the gut bacteria that impact composition of the bile acid pool, which has implications in the context of metabolic disorders and cancers of the digestive tract. Our results greatly expand upon the list of bacterial species known to perform different types of bile acid transformations. This knowledge will be vital for assessing the causal connections between the microbiome, bile acid pool composition, and human health.

Keywords: bile acids; conjugation; gut bacteria; mass spectrometry; microbially conjugated bile acids; microbiome.

FIG 1

Bile acid production and enterohepatic circulation. Conjugated bile acids are synthesized from cholesterol in the liver and stored in bile in the gallbladder. After being released from the gallbladder into the duodenum, bile acids travel through the jejunum and into the ileum. Ninety-five percent of bile acids are actively absorbed from the small intestine and returned to the liver via enterohepatic circulation. The remaining 5% that reach the large intestine can be passively reabsorbed or deconjugated and transformed into secondary bile acids by the gut microbiota. Bacterial transformation of bile acids can also occur in the small intestine to a lesser extent. Secondary bile acids can be excreted, or they can be absorbed in the large intestine to join enterohepatic circulation. This figure shows primary bile acids conjugated to glycine moieties.

FIG 2

Secondary bile acid production observed in vitro. (A) Numbering of carbon atoms on the bile acid steroid core. (B) 7α-dehydrogenation of CDCA and CA. (C) 12α-dehydrogenation of CA and DCA. (D) 3α-dehydrogenation of CA, CDCA, and DCA. (E) Transformation of CA into 7,12-dioxoLCA. (F) 7α-dehydroxylation of CDCA and CA. (G) Other transformations. CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; HDCA, hyodeoxycholic acid; LCA, lithocholic acid; UDCA, ursodeoxycholic acid; β-MCA, β-muricholic acid; 3-oxoCA, 3-oxocholic acid; 3-oxoCDCA, 3-oxochenodeoxycholic acid; 3-oxoDCA, 3-oxodeoxycholic acid; 7-oxoDCA, 7-oxodeoxycholic acid; 7-oxoLCA, 7-oxolithocholic acid; 12-oxoCDCA, 12-oxochenodeoxycholic acid; 12-oxoLCA, 12-oxolithocholic acid; 7,12-dioxoLCA, 7,12-dioxolithocholic acid. A complete list of bile acids and abbreviations used in this study can be found in Table S2.

FIG 3

Secondary bile acid production across bacterial strains. The heat map shows the bile acid steroid core transformations carried out by each isolate. The color scale denotes amounts of secondary bile acid produced at 24 (left) and 48 h (right). Bacteria were provided with 100 μM cholic acid (CA), chenodeoxycholic acid (CDCA), or deoxycholic acid (DCA). The detection limit was below 0.05 μM for all bile acids. Phyla information is indicated by color-coded dashed lines in the phylogenetic tree. Heat maps for bile acid production when CA, CDCA, and DCA were added in combination are shown in Fig. S2.

FIG 4

Relative activity and specificity of α-dehydrogenase activity. (A) 3α-, 7α-, and 12α-dehydrogenation activity when bile acids were administered separately. The median production of oxo bile acids is denoted by a solid colored line. Data shown are the average of 24 and 48 h measurements. (B) For each isolate capable of 3α-dehydrogenation, the amounts of CDCA, CA, and DCA transformed were plotted against each other. For each isolate capable of 7α-dehydrogenation, the amounts of CA and CDCA transformed were plotted against each other. For each isolate capable of 12α-dehydrogenation, the amounts of CA and DCA transformed were plotted against each other. Data shown are the averages of 24 and 48 h measurements.

FIG 5

Production of amino acid-conjugated bile acids across bacterial strains. (A) The heat map shows the bile acid-to-amino acid conjugations carried out by each isolate at 24 h. The data are presented as raw signal intensities normalized by Z-score, which is denoted by the color scale. Bacteria were provided with 100 μM cholic acid (CA), chenodeoxycholic acid (CDCA), or deoxycholic acid (DCA). Phyla information is indicated by the color-coded dashed lines in the phylogenetic tree on the left. The heat map for conjugated bile acid production at 48 h is shown in Fig. S3. (B) MS/MS spectra of selected novel microbially conjugated bile acids. Parent ion structure, mass, and retention time are listed along with the structure and exact masses for three identifying fragments: the major sterol fragment, the fragment resulting from amino acid loss, and the amino acid fragment.