Bile Salt Hydrolases with Extended Substrate Specificity Confer a High Level of Resistance to Bile Toxicity on Atopobiaceae Bacteria

Abstract

The bile resistance of intestinal bacteria is among the key factors responsible for their successful colonization of and survival in the mammalian gastrointestinal tract. In this study, we demonstrated that lactate-producing Atopobiaceae bacteria (Leptogranulimonas caecicola TOC12^T and Granulimonas faecalis OPF53^T) isolated from mouse intestine showed high resistance to mammalian bile extracts, due to significant bile salt hydrolase (BSH) activity. We further succeeded in isolating BSH proteins (designated LcBSH and GfBSH) from L. caecicola TOC12^T and G. faecalis OPF53^T, respectively, and characterized their enzymatic features. Interestingly, recombinant LcBSH and GfBSH proteins exhibited BSH activity against 12 conjugated bile salts, indicating that LcBSH and GfBSH have much broader substrate specificity than the previously identified BSHs from lactic acid bacteria, which are generally known to hydrolyze six bile salt isomers. Phylogenetic analysis showed that LcBSH and GfBSH had no affinities with any known BSH subgroup and constituted a new BSH subgroup in the phylogeny. In summary, we discovered functional BSHs with broad substrate specificity from Atopobiaceae bacteria and demonstrated that these BSH enzymes confer bile resistance to L. caecicola TOC12^T and G. faecalis OPF53^T.

Keywords: Granulimonas faecalis; Leptogranulimonas caecicola; bile resistance; bile salt hydrolase; probiotics.

Figure 1

Bile salt hydrolase (BSH) activity in Leptogranulimonas caecicola TOC12^T and Granulimonas faecalis OPF53^T. Full-grown cultures were streaked on a Gifu anaerobic medium (GAM) agar plate (left) and a GAM agar plate supplemented with 0.1% glycodeoxycholic acid (right). All plates were anaerobically incubated at 37 °C for 5 days. The visible precipitates surrounding colonies are the well-known indicators of bacterial BSH activity [12,27].

Figure 2

Sequence analyses of the putative LcBSH and GfBSH. (A) Overall and (B) catalytic active site superposition images of LcBSH (blue) and GfBSH (green) with CpBSH from Clostridium perfringens (gray; PDB accession number 2RLC). The degradation products (glycine and cholic acid) of glycocholic acid by CpBSH are shown in magenta sticks. (C) Multiple alignment analysis of the putative LcBSH and GfBSH. Amino acid sequences of LcBSH and GfBSH were compared with characterized bile salt hydrolases (BSHs) from gut bacteria. The black and gray backgrounds indicate identical and similar amino acid residues, respectively. The conserved residues (Cys, Arg, Asp, Asn, and Arg) related to the catalytic active site are boxed in red lines. Abbreviated as: BlBSH (AAF67801) from Bifidobacterium longum SBT2928; CpBSH (P54965) from Clostridium perfringens 13; LgBSH (WP_020806888) from Lactobacillus gasseri FR4.

Figure 3

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified LcBSH and GfBSH. The purified proteins were loaded onto 12% SDS-PAGE gels. The single protein bands of purified His₆-LcBSH and His₆-GfBSH proteins were observed to be around 35 kDa. Lane M, molecular size-marker (3-color prestained XL-Ladder, APRO Science, Tokushima, Japan).

Figure 4

Bile salt hydrolase (BSH) activity and biochemical characterization of recombinant LcBSH and GfBSH proteins. (A) BSH activity and substrate specificity of LcBSH and GfBSH. The tested substrates were glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycoursodeoxycholic acid (GUDCA), glycohyodeoxycholic acid (GHDCA), glycolithocholic acid (GLCA), taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), taurohyodeoxycholic acid (THDCA), and taurolithocholic acid (TLCA). Values represent the mean of three independent experiments (each n = 3). Error bars represent standard deviation (SD). (B) Effect of temperature (20–80 °C) and (C) pH (pH 3.0–pH 10.0) on BSH activity of LcBSH and GfBSH. Each value represents the mean of six technical replicates (each n = 6). Maximum activity was defined as 100%. Error bars indicate SD.

Figure 5

Phylogenetic analysis of LcBSH and GfBSH. Amino acid sequences of LcBSH and GfBSH proteins were aligned with previously identified bile salt hydrolases (BSHs). The phylogenetic tree was constructed with MEGA X software, using the neighbor-joining method (1000 bootstrap replications) [52]. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The phylogenetic tree was displayed and customized using the online tool Interactive Tree Of Life (iTOL v6) [53]. Bootstrap values are shown by circle symbols whose size correlates with the bootstrap values. BsPVA from Lysinibacillus sphaericus was used as an outgroup.