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. 2021 Feb 9;118(6):e2017709118.
doi: 10.1073/pnas.2017709118.

Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization

Matthew H Foley  1 , Sarah O'Flaherty  2 , Garrison Allen  1 , Alissa J Rivera  1 , Allison K Stewart  3 , Rodolphe Barrangou  4 , Casey M Theriot  5
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V体育官网入口 - Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization

Matthew H Foley et al. Proc Natl Acad Sci U S A. .

Abstract

Primary bile acids (BAs) are a collection of host-synthesized metabolites that shape physiology and metabolism. BAs transit the gastrointestinal tract and are subjected to a variety of chemical transformations encoded by indigenous bacteria. The resulting microbiota-derived BA pool is a mediator of host-microbiota interactions VSports手机版. Bacterial bile salt hydrolases (BSHs) cleave the conjugated glycine or taurine from BAs, an essential upstream step for the production of deconjugated and secondary BAs. Probiotic lactobacilli harbor a considerable number and diversity of BSHs; however, their contribution to Lactobacillus fitness and colonization remains poorly understood. Here, we define and compare the functions of multiple BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri Our genetic and biochemical characterization of lactobacilli BSHs lend to a model of Lactobacillus adaptation to the gut. These findings deviate from previous notions that BSHs generally promote colonization and detoxify bile. Rather, we show that BSH enzymatic preferences and the intrinsic chemical features of various BAs determine the toxicity of these molecules during Lactobacillus growth. BSHs were able to alter the Lactobacillus transcriptome in a BA-dependent manner. Finally, BSHs were able to dictate differences in bacterial competition in vitro and in vivo, defining their impact on BSH-encoding bacteria within the greater gastrointestinal tract ecosystem. This work emphasizes the importance of considering the enzymatic preferences of BSHs alongside the conjugated/deconjugated BA-bacterial interaction. These results deepen our understanding of the BA-microbiome axis and provide a framework to engineer lactobacilli with improved bile resistance and use probiotics as BA-altering therapeutics. .

Keywords: Acidophilus; Lactobacillus; bile acid; bile salt hydrolase; gasseri V体育安卓版. .

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V体育ios版 - Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
BA structures differ in inhibition of Lactobacillus species. (A) BA structures, abbreviations used in this study. All structures shown in SI Appendix, Fig. S1. CMC values calculated in SI Appendix, Fig. S2 and represent the mean CMC from n = 2 experiments ± SEM. (B) WT L. acidophilus (La WT), ΔbshAB L. acidophilus (La ΔbshAB), WT L. gasseri (Lg WT), and ΔbshAB L. gasseri (Lg ΔbshAB) monocolonization of n = 5 to 8 germ-free C57BL/6 mice. CFUs were counted from cecal contents 7 d after colonization. Asterisks represent significant (*P < 0.05) differences from WT by Mann–Whitney U test. (C) BSH-null L. acidophilus and L. gasseri (ΔbshAB) strains were used to determine BA MICs. Bars represent mean MICs from n = 3 independent experiments. MICs did not vary between experiments.
Fig. 2.
Fig. 2.
BSHs impact Lactobacillus fitness in a genotypic and bile acid specific-manner. (A) L. acidophilus and (B) L. gasseri BSH mutants grown for 24 h in MRS, 5 mM GCA, 5 mM TCA, 2.5 mM GCDCA, 2.5 mM TCDCA, 2.5 (A)/1.25 (B) mM GDCA, or 5 mM TDCA. Error bars represent SD from n = 4 independent experiments. Dashed lined denotes the approximate starting CFUs/mL at 0 h. Asterisks represent significant (*P < 0.05) differences from WT by Mann–Whitney U test.
Fig. 3.
Fig. 3.
Lactobacillus BSHs display variable preferences for bile acid conjugation. Average specific activities from (A) LaBSHa, (B) LaBSHb, (C) LgBSHa, and (D) LgBSHb were determined by the Ninhydrin assay on the same panel of conjugated BAs used in Fig. 2 supplemented with glycoursodeoxycholic acid (GUDCA) and taurolithocholic acid (TLCA). Error bars represent SD from n = 3 independent experiments.
Fig. 4.
Fig. 4.
BSH activity alters membrane integrity and global transcriptome in a BA-specific manner. (A and B) PI staining to assess membrane integrity of midlog (OD 600 nm = 0.7) grown Lactobacillus exposed to various BAs or heat killed (HK). BA concentrations were the same as those used in Fig. 2. Normalized fluorescence was calculated by subtracting background PI fluorescence and normalizing to the starting OD 600 nm at BA exposure. Bars represent average fluorescence from n = 3 independent experiments and error bars represent SD. Asterisks represent significant (*P < 0.05) differences between groups by Welch’s t test.
Fig. 5.
Fig. 5.
BSH activity drives competitive dynamics in vitro and in vivo. (A and B) CIs for Lactobacillus cocultures anaerobically for 24 h in the presence of various BAs. BA concentrations were the same as those used as in Fig. 2. CIs were calculated as follows: CI = Final[Log10(ΔbshAB CFUs)/Log10(WT CFUs)]/Initial[Log10(ΔbshAB CFUs)/Log10(WT CFUs)]. Dashed lines denote a CI = 0. Asterisks represent significant (P < 0.05) differences between groups by Mann–Whitney U test. (C) L. acidophilus and L. gasseri WT/ΔbshAB cocolonization of n = 6 germ-free C57BL/6 mice. CFUs were counted from cecal contents 7 d after colonization. Asterisks represent significant (P < 0.05) differences from WT by Mann–Whitney U test. (D) Cocolonization of n = 3 cecal contents from conventional C57BL/6 mice. Asterisks represent significant (*P < 0.05) differences from WT by unpaired t test.
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