Identification of Bile Salt Hydrolase and Bile Salt Resistance in a Probiotic Bacterium Lactobacillus gasseri JCM1131T

Abstract

Lactobacillus gasseri is one of the most likely probiotic candidates among many Lactobacillus species. Although bile salt resistance has been defined as an important criterion for selection of probiotic candidates since it allows probiotic bacteria to survive in the gut, both its capability and its related enzyme, bile salt hydrolase (BSH), in L. gasseri is still largely unknown. Here, we report that the well-known probiotic bacterium L. gasseri JCM1131^T possesses BSH activity and bile salt resistance capability. Indeed, this strain apparently showed BSH activity on the plate assay and highly tolerated the primary bile salts and even taurine-conjugated secondary bile salt. We further isolated a putative BSH enzyme (LagBSH) from strain JCM1131^T and characterized the enzymatic function. The purified LagBSH protein exhibited quite high deconjugation activity for taurocholic acid and taurochenodeoxycholic acid. The lagBSH gene was constitutively expressed in strain JCM1131^T, suggesting that LagBSH likely contributes to bile salt resistance of the strain and may be associated with survival capability of strain JCM1131^T within the human intestine by bile detoxification. Thus, this study first demonstrated the bile salt resistance and its responsible enzyme (BSH) activity in strain JCM1131^T, which further supports the importance of the typical lactic acid bacterium as probiotics.

Keywords: Lactobacillus gasseri; Ntn-hydrolase family protein; bile salt hydrolase; probiotics.

Figure 1

Bile salt hydrolase activity in Lactobacillus gasseri JCM1131^T. Full-grown culture of L. gasseri JCM1131^T was streaked on an MRS agar plate (A) or an MRS agar plate supplemented with 0.25% taurodeoxycholic acid (B). The plates were anaerobically incubated at 37 °C for 5 days. The visible halo surrounding colonies and the white precipitates with colonies are the well-known indicator of bacterial BSH activity [19,30].

Figure 2

Bile salt tolerance activity in Lactobacillus gasseri JCM1131^T. Full-grown culture of strain JCM1131^T was mixed with GCA, GDCA, TCA, and TDCA at final concentrations of 0.05% and incubated anaerobically at 37 °C. The optimal density (OD₆₀₀) was measured every hour and survival rates were calculated as described previously [26]. The survival rate of control (without bile salt) was defined as 100%. Results indicated mean ± SD obtained in triplicate experiments.

Figure 3

(A) Physical map of the predicted bsh gene on the genome sequence of Lactobacillus gasseri JCM1131^T (accession number CP000413). The scale bar indicates a 1 kb length of nucleotide. A putative bsh gene (lagBSH) and its surrounding ORFs are represented by filled and open symbols, respectively. Brief annotation and protein ID were provided. MegG, demethylmenaquinone methyltransferase; MP, membrane protein. (B) Multiple alignment of amino acid sequences of BSHs. Amino acid sequence of LagBSH was aligned and compared with known BSHs from Lactobacillus species. The black and gray shading indicates identical and similar amino acid residues, respectively. The conserved residues (Cys, Arg, Asp, Asn, and Arg) relevant to the predicted active site are indicated by black asterisks. Abbreviations: LaBSH (AAV42923) from Lactobacillus acidophilus NCFM; LgBSH (WP_020806888) from Lactobacillus gasseri FR4; LjBSH (AAC34381) from Lactobacillus johnsonii 100-100; LsBSH (JX120368) from Lactobacillus salivarius B-30514.

Figure 4

Phylogenetic analysis of LagBSH with cholylglycine hydrolase family proteins. The phylogenetic tree was constructed with MEGA X software using the neighbor-joining method (1000 bootstrap replications) [39]. Bootstrap values greater than 50% are shown by circle symbols whose size correlates with the bootstrap values. CpBSH, BSH from Clostridium perfringens 13, was used as an outgroup.

Figure 5

Bile salt hydrolase activity and biochemical characterization of LagBSH. (A) BSH activities were measured toward five human bile salts: glycocholicacid (GCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), taurodeoxycholic acid (TDCA), and taurochenodeoxycholic acid (TCDCA). Values are indicated as means for eight technical experiments (n = 8). Error bars represent standard deviation (SD). (B) Effect of temperature (10–90 °C) and (C) pH (pH 3.0–pH 10.0) on BSH activity toward TCA of LagBSH. Each value is expressed as means for eight technical replicates (n = 8). Maximum activity was taken as 100%.

Figure 6

RT-PCR analyses of lagBSH genes in L. gasseri JCM1131^T. Sterilized water (lane N) and genomic DNA of strain JCM1131^T (lane P) were used as negative and positive control, respectively. The products of reverse transcription from total RNA of nonsupplemented (lane 1), TCA-supplemented (lane 2), and TDCA-supplemented (lane 3) strain JCM1131^T cells were used as the template for PCR, respectively. The 16S rRNA gene (378 bp) was used as internal standard control. Lane M, molecular size markers (100 bp DNA ladder, Promega, Madison, WI, USA).