Taxonomic identification of bile salt hydrolase-encoding lactobacilli: Modulation of the enterohepatic bile acid profile

Abstract

Bile salt hydrolases (BSHs) are enzymes that are essential for the enterohepatic metabolism of bile acids (BAs). BSHs catalyze the production of unconjugated BAs and regulate the homeostasis of BA pool. This study identified Lactobacillus as a crucial BSH-encoding genus, and 16 main species were obtained using metagenomic data from publicly available human gut microbiome databases. Then, the 16 species of lactobacilli were classified into four typical categories by BSH phylotypes, including five species encoding BSH-T0, six species encoding BSH-T2, four species encoding BSH-T3, and Ligilactobacillus salivarius encoding both BSH-T0 and BSH-T3. The lactobacilli with the highest in vitro deconjugation activities against seven conjugated BAs were the BSH-T3-encoding strains. Furthermore, in vivo studies in mice administered four representative lactobacilli strains encoding different BSH phylotypes showed that treatment with BSH-T3-encoding Limosilactobacillus reuteri altered the structure of the gut microbiome and metabolome and significantly increased the levels of unconjugated BAs and total BA excretion. Our findings facilitated the taxonomic identification of crucial BSH-encoding lactobacilli in human gut microbiota and shed light on their contributions toward modulation of the enterohepatic circulation of BAs, which will contribute to future therapeutic applications of BSH-encoding probiotics to improve human health.

Keywords: bile acid; bile salt hydrolase; gut microbiota; lactobacilli; phylotype.

Figure 1

Comprehensive analysis of bile salt hydrolase (BSH)‐encoding bacteria. (A) Phylogenetic tree of 156 BSHs, accompanied by their relative abundance in the human microbiome. Columns with the same color represent BSHs from the same genus. In the same BSH phylotype, the lighter the color of the column, the lower the proportion of the total relative abundance of BSHs encoded by the genus. Genera with abundance of less than 5% are represented by gray columns. (B) Multiple factor analysis of representative BSH‐encoding genera. The diversity is represented by the Shannon index, the deconjugation ability is defined as the proportion of 100 μM conjugated bile salts hydrolyzed to unconjugated bile salt by BSHs after 48 h of reaction (detected by liquid chromatography triple quadrupole mass spectrometry), and the specific enzymatic activity is determined by the amount of BSHs that release 1 μmol glycine or taurine from 20 mM conjugated bile salts per minute in ninhydrin assay. The red columns represent the top genera during the analysis of the corresponding factor. (C) Radar analysis of representative BSH‐encoding genera; the data for each factor were normalized. (D) Relative abundance of BSH‐encoding lactobacilli strains in different populations.

Figure 2

Deconjugation activity of bile salt hydrolase (BSH)‐encoding lactobacilli. (A) Taxonomic characteristics of representative BSH‐encoding lactobacilli strains. Details are shown in Supporting Information: Table S6. (B) In vitro deconjugation activity of representative BSH‐encoding lactobacilli strains. Details are shown in Supporting Information: Table S7. *p < 0.05, **p < 0.01, and ***p < 0.001. In this experiment, unconjugated BAs include CA, CDCA, DCA, and UDCA. DCA and UDCA belong to secondary BAs, which also could transfer from CA and CDCA. CA, Cholic acid; CDCA, Chenodeoxycholic acid; DCA, Deoxycholic acid; GCA, Sodium glycocholate hydrate; GCDCA, sodium glycochenodeoxycholate; GDCA, sodium glycodeoxycholate; TCA, sodium taurocholate hydrate; TCDCA, sodium taurochnodeoxycholate; TDCA, sodium taurodeoxycholate hydrate; TUDCA, sodium tauroursodeoxy cholate; UDCA, Ursodeoxycholic acid.

Figure 3

Treatment with lactobacilli alters the structure of the fecal microbiome in mice. (A) Alpha diversity (Chao richness and Shannon diversity index) of the fecal microbiome. (B) Two‐dimensional PCoA of the fecal microbiome based on WUniFrac. PERMANOVA was used for statistical significance of beta diversity; p < 0.05 was considered as statistically significant. (C) Volcano plots illustrate the genus enrichment analysis of the fecal microbiome. The Wilcoxon rank sum test was used for analysis. The labels represent the top 15 genera, and the sizes of labels and points were proportional to the average abundance of genera in each group. (D) Concentrations of four lactobacilli species (including Limosilactobacillus fermentum, Ligilactobacillus animalis, Ligilactobacillus salivarius, and Limosilactobacillus reuteri) in the fecal microbiota of mice in different groups. PCoA, principal coordinates analysis. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4

Treatment with lactobacilli alters the serum and fecal metabolomes of mice. (A) Heatmap of differential metabolic ion enrichment analysis in serum. The red color shows that the relative concentration of the feature was higher in the lactobacilli‐administered group, while the blue color shows that the relative concentration of the feature was higher in the control group; the gray color shows that the relative concentration of the feature did not differ between the two groups. (B) Score plots of principal component analysis based on the differential metabolites (VIP > 1, pFDR ≤ 0.05) in serum. (C) Heatmap of the differential metabolites (pFDR ≤ 0.05) in serum. (D) Heatmap of differential metabolic ion enrichment analysis in feces. (E) Score plots of principal component analysis based on the differential metabolites (VIP > 1, pFDR ≤ 0.05) in feces. (F) Heatmap of the differential metabolites (pFDR ≤ 0.05) in feces. Asterisks represent differential metabolites in the L. reuteri‐administered group. Positively charged ions are collected in ESI+ mode, and negatively charged ions are collected in ESI− mode.

Figure 5

Treatment with lactobacilli alters the composition of the bile acid (BA) pool in mice. (A) Unconjugated BAs. (B) Total BA. (C) Proportion of unconjugated BAs and conjugated BAs in the liver of mice from different groups. (D) Unconjugated BAs. (E) Total BA. (F) Proportion of unconjugated BAs and conjugated BAs in the serum of mice from different groups. (G) Unconjugated BAs. (H) Total BA. (I) Proportion of unconjugated BAs and conjugated BAs in the distal ileum of mice from different groups. (J) Unconjugated BAs. (K) Total BA. (L) Proportion of unconjugated BAs and conjugated BAs in the fecal samples of mice from different groups. Statistically significant differences between the two groups were determined using a paired t test. *p < 0.05, **p < 0.01, and ***p < 0.001. Unconjugated BAs include CA, CDCA, DCA, and UDCA. Conjugated BAs include GCA, GCDCA, GDCA, TCA, TCDCA, TDCA, and TUDCA. Total BAs are the sum of all the detected BAs (including unconjugated BAs and conjugated BAs). CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; F, female; LCA, lithocholic acid; M, male; UDCA, ursodeoxycholic acid; β‐MCA, β‐Muricholic acid.