Identification and Characterization of Major Bile Acid 7α-Dehydroxylating Bacteria in the Human Gut

Abstract

The metabolism of bile acids (BAs) by gut bacteria plays an important role in human health. This study identified and characterized 7α-dehydroxylating bacteria, which are majorly responsible for converting primary BAs to secondary BAs, in the human gut and investigated their association with human disease. Six 7α-dehydratase (BaiE) clusters were identified from human gut metagenomes through sequence similarity network and genome neighborhood network analyses. Abundance analyses of gut metagenomes and metatranscriptomes identified a cluster of bacteria (cluster 1) harboring baiE genes that may be key 7α-dehydroxylating bacteria in the human gut. The baiE gene abundance of cluster 1 was significantly and positively correlated with the ratio of secondary BAs to primary BAs. Furthermore, the baiE gene abundances of cluster 1 were significantly negatively correlated with inflammatory bowel disease, including Crohn's disease and ulcerative colitis, as well as advanced nonalcoholic fatty liver disease, liver cirrhosis, and ankylosing spondylitis. Phylogenetic and metagenome-assembled genome analyses showed that the 7α-dehydroxylating bacterial clade of cluster 1 was affiliated with the family Oscillospiraceae and may demonstrate efficient BA dehydroxylation ability by harboring both a complete bai operon, for proteins which produce secondary BAs from primary BAs, and a gene for bile salt hydrolase, which deconjugates BAs, in the human gut. IMPORTANCE In this study, we identified a key 7α-dehydroxylating bacterial group predicted to be largely responsible for converting primary bile acids (BAs) to secondary BAs in the human gut through sequence similarity network, genome neighborhood network, and gene abundance analyses using human gut metagenomes. The key bacterial group was phylogenetically quite different from known 7α-dehydroxylating bacteria, and their abundance was highly correlated with the occurrence of diverse diseases associated with bile acid 7α-dehydroxylation. In addition, we characterized the metabolic features of the key bacterial group using their metagenome-assembled genomes. This approach is useful to identify and characterize key gut bacteria highly associated with human health and diseases.

Keywords: 7α-dehydratase; BaiE; IBD; bile acids; human gut microbiota; metabolic pathways; sequence similarity networks.

FIG 1

Sequence similarity networks (SSNs) of 135 protein sequences (green) identified from human gut microbiome whole-genome sequences, based on an alignment score of 70 (approximately 90% sequence identity). The BaiE sequences that had been previously experimentally verified (blue) or obtained from the UniRef100 database (red) were also used to generate the SSNs. Each node in the SSNs represents a protein, and alignment scores between proteins in different clusters are <70. Proteins within the same clusters have sequence identities of >92.4%. Numbers reflect arbitrary naming of clusters.

FIG 2

Phylogenetic tree of putative BaiE sequences based on the maximum-likelihood algorithm. Names of strains harboring putative BaiE sequences are in parentheses, and the superscript “U” indicates uncultured strains. Organisms whose BaiE sequences were previously experimentally verified are in bold. Bootstrap values of >70% are shown on the branches as percentages of 1,000 replicates.

FIG 3

Gene neighborhood analysis of putative baiE genes present in the bai operons of representative genomes of each phylogenetic cluster (Fig. 2). Asterisks indicate gene structures of bai operons that were previously experimentally verified to be able to convert primary bile acids to secondary bile acids.

FIG 4

Confirmation of enzymatic functions of the putative baiA (DER43_01330) and baiB (DER43_01335) genes in the uncultured Clostridiales bacterium UBA11811. A diagram showing the enzymatic reactions of cholic acid by BaiA and BaiB proteins (A). LC-Q-TOF-MS ion chromatograms showing the production of cholyl-CoA (m/z 1158.4026 of [M+H]⁺, 6.544 min) by BaiB (B) and 3-oxo-cholyl-CoA (m/z 1156.3852 of [M+H]⁺, 6.428 min) by BaiB and BaiA (C) from cholic acid. Cell lysate of E. coli BL21(DE3) harboring the pET28a plasmid without a DNA insert was used as a negative control.

FIG 5

Relative abundances of baiE genes (A) and baiE gene transcripts (B) of BaiE clusters in the human gut microbiota. The abundance of baiE genes and baiE gene transcripts in each sample was normalized based on read counts per million sequencing reads. Pink circles indicate the mean values of the relative abundances of baiE genes or baiE gene transcripts in each cluster. Mean values, with percentages of total counts in parentheses, are also indicated above the data.

FIG 6

Relative abundance of baiE genes of BaiE clusters 1 (A), 4 (B), and 6 (C) according to disease cases in human gut metagenomes. *, P < 0.05; **, P < 0.01; ***, P < 0.001. CD, Crohn’s disease; UC, ulcerative colitis; NAFLD, nonalcoholic fatty liver disease; LC, liver cirrhosis; CRC, colorectal cancer; ACVD, atherosclerotic cardiovascular disease; AS, ankylosing spondylitis.

FIG 7

Spearman correlations between baiE abundances of all sequences (A), cluster 1 (B), cluster 4 (C), and cluster 6 (D) and ratios of secondary BAs to primary BAs in the human gut. Each dot represents the results from a participant. The r and p values are the Spearman correlation and significant coefficients, respectively.

FIG 8

Neighbor-joining tree based on 120 concatenated marker proteins, showing the phylogenetic positions of 41 MAGs harboring BaiE genes of cluster 1. The tree consists of genomes of all cultured type strains belonging to the class Clostridia, except for the 41 MAGs. Colored circles indicate the possible presence of genes associated with 7α-dehydroxylation of primary BAs, and asterisks indicate genomes capable of converting primary BAs to secondary BAs by harboring a bai operon. The bar indicates the number of substitutions per site.

FIG 9

Proposed metabolic pathways for carbohydrates, bile acids, vitamins, cofactors, amino acids, and polyamines and several systems of human gut MAG bacteria of cluster 1 harboring a bai operon. Major metabolic pathways and systems are distinguished by shading colors, and unidentified but likely metabolic pathways are indicated with dotted blue arrows.