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Bile salt hydrolase acyltransferase activity expands bile acid diversity

Nature volume 626, pages 852–858 (2024)Cite this article

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Abstract

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1,2,3,4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

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Fig. 1: C. perfringens BSH/T produces a broad range of MCBAs at acidic pH.
Fig. 2: MCBA product identities correlate with BSH/T amino acid sequences.
Fig. 3: MCBAs show varied antimicrobial properties.
Fig. 4: BA concentrations in mouse tissue samples following MCBA feeding and in human faeces of patients undergoing sleeve gastrectomy.

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Data availability

Protein structures are available on the Protein Data Bank. C. perfringens BSH/T in complex with DCA and taurine, from refs. 17,19, is available under PDB ID 2BJG (https://doi.org/10.2210/pdb2BJG/pdb). L. salivarius BSH/T in complex with TCA, from refs. 21,40, is available under PDB ID 8BLT (https://doi.org/10.2210/pdb8blt/pdb). Raw mass spectrometry data are publicly available in the MassIVE database (massive.ucsd.edu) for the in vitro screen for MCBA production under MSV000090234 (https://doi.org/10.25345/C5S756Q1B), for CpBSH/T variant analysis under MSV000092138 (https://doi.org/10.25345/C55D8NQ9V), for MCBA gavage samples under MSV000093173 (https://doi.org/10.25345/C57S7J35N), for mixed MCBA PBFM dosing under MSV000093171 (https://doi.org/10.25345/C5H98ZQ3R), for 100 mg−1 kg of SerCA PBFM dosing at MSV000093169 (https://doi.org/10.25345/C5RV0DB2C), 10 mg−1 kg of MCBA PBFM dosing under MSV000093172 (https://doi.org/10.25345/C5CJ87W9C) and for SG faecal samples under MSV000093167 (https://doi.org/10.25345/C51834C9N). GNPS molecular networks are available for the MCBA production screen at gnps.ucsd.edu/ProteoSAFe/status.jsp?task=565151309a874d5f97caa3f383c95382, for CpBSH/T incubation with 1 mM BA and equimolar amino acid mix at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=3dec8f7ab26d47098406a7e597825154 and https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=33da5da024ed44848770a4a02b119d9e, for the CpBSH/T mutagenesis experiment at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=30c88ca297a44f84be5fa32b376e5cb9 and for the SG faecal samples at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f11eaab1cf1d43b1a5f754575d171e87. 16S rRNA gene amplicon data were deposited in the EMBL-EBI European Nucleotide Archive. Data from the 100 mg kg−1 gavage experiment can be found under project PRJEB68000, study accession ERP153011. Tissue data from the 10 mg kg−1 PBFM experiment can be found under project PRJEB68146, study accession ERP153132. Faecal data available from 10 mg kg−1 PBFM experiment are available under project PRJEB68149, study accession ERP153135. Analyses can be found on Qiita under analysis ID 53128 for the 100 mg kg−1 gavage and IDs 57407 and 57481 for tissue and faecal samples, respectively, from the 10 mg kg−1 PBFM experiment.  Source data are provided with this paper.

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Acknowledgements

We thank C. M. Waters, N. D. Hammer and K. Parent for generously providing some of the strains used in this work in addition to P. Lawson for his help and input acquiring strains from the Culture Collection, University of Gothenburg. We would also like to thank J. B. Gomez, E. N. Ottosen and K. C. Ford for their guidance. This work was funded by Michigan State University and the Global Grants for Gut Health, cosponsored by Yakult and Nature Research.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, USA

    Douglas V. Guzior, Maxwell Okros, Madison Shivel, Bruin Armwald, Christopher Bridges, Yousi Fu, Christian Martin, Robert P. Hausinger & Robert A. Quinn

  2. Department of Microbiology, Genetics & Immunology, Michigan State University, East Lansing, MI, USA

    Douglas V. Guzior, Christopher Bridges & Robert P. Hausinger

  3. College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA

    Madison Shivel & Bruin Armwald

  4. Mass Spectrometry and Metabolomics Core, Michigan State University, East Lansing, MI, USA

    Anthony L. Schilmiller

  5. Corewell Health, William Beaumont University Hospital, Royal Oak, MI, USA

    Wendy M. Miller, Kathryn M. Ziegler, Matthew D. Sims, Michael E. Maddens & Stewart F. Graham

  6. Oakland University, William Beaumont School of Medicine, Rochester, MI, USA

    Wendy M. Miller, Kathryn M. Ziegler, Matthew D. Sims, Michael E. Maddens & Stewart F. Graham

  7. Beaumont Research Institute, Royal Oak, MI, USA

    Stewart F. Graham

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  1. Douglas V. Guzior
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Contributions

D.V.G., S.F.G. and R.A.Q. designed the project. D.V.G. and R.A.Q. discovered acyl transfer activity by bile salt hydrolase and performed phylogenetic analysis. D.V.G. generated data for in vitro BSH/T characterization, species-specific MCBA production screens, MCBA antimicrobial activity and heterologous bsh/t expression experiments. D.V.G. and C.B. generated data for pancreatic carboxypeptidase activity. M.O. raised mice. M.O., M.S. and B.A. conducted work with animals. D.V.G., M.O., M.S., B.A., C.B. and Y.F. collected samples and generated data from animal experiments. W.M.M., K.M.Z., M.D.S. and M.E.M. coordinated human sample collection, treated patients, performed bariatric surgeries and completed all clinical follow-up. D.V.G., C.M. and R.A.Q. analysed data. M.O., A.L.S., S.F.G., R.P.H. and R.A.Q. guided experimental design and analysis. D.V.G. and R.A.Q. wrote the manuscript.

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Correspondence to Robert A. Quinn.

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Extended data figures and tables

Extended Data Fig. 1 MCBA abundance following purified CpBSH/T or microbial incubation with BAs.

a, Summed MCBA auc after 120 min incubation of CpBSH/T, 2.5 mM TCA and 125 µM equimolar amino acid mix at various pH revealing pH-dependence of BA conjugation, n = 3 independent reactions. Red dashed line indicates the pH 5.3 optimum following derivation as determined by fitting a 5-factor polynomial equation. b, Proportion of TCA and CA in the bile acid pool when CpBSH/T was incubated with 8 mM TCA at different pH values across time. n = 3 replicates. c, Goodness of fit outputs for curve fitting to determine CpBSH/T pH optimum, values in parentheses as sem. Equation used to calculate pH optimum (5-factor, bolded) based on adjusted R2. Coefficient significance determined by one-sided t test and model significance determined by one-way ANOVA without P value adjustment. *P < 0.1, **P < 0.05 ***P < 0.01. d, Relative abundances of MCBAs produced by purified CpBSH/T. Enzyme was individually provided 1 mM BA and an equimolar mix of amino acids, buffered at pH 5, and sampled after 120-min incubation at 37 °C. Data presented as mean auc abundance (sem), n = 3. e, Nonmetric data scaling using Bray-Curtis dissimilarity of amino acids used in BA conjugation, using average amino acid auc per strain. Colour represents cluster assigned based on cluster analysis and dot size represents the average total MCBA abundance. n = 3 independent cultures. f, CpBSH/T (PBD ID: 2bjg)17,19 cocrystalized with TDCA and residues important for BA deconjugation are highlighted in addition to Asn82, the residue playing a key role in BA reconjugation specificity.

Source Data

Extended Data Fig. 2 Clostridium scindens genome analysis for putative bsh/t annotation.

a, Phylogenetic analysis of 35 publicly available genomes for C. scindens. The ATCC type strain, used in this work, has two deposited genomes and is highlighted in red. The only strain with predicted bsh/t was C. scindens strain Q4, highlighted in blue. b, Pairwise BSH/T amino acid sequence similarity of all strains included in this work (matching Fig. 2c), now including the predicted BSH/T present in C. scindens strain Q4 (NZ_CP080442.1_958, based on Prokka analysis).

Extended Data Fig. 3 GCA and TCA extracted ion chromatograms following 24 h induction of C. perfringens BSH/T variants in E. coli.

Representative a, GCA and b, TCA extracted ion chromatograms showing significantly diminished in WT and N82Y variant strains with minimal change in the C2A variant and EV control.

Extended Data Fig. 4 Amino acid-dependency of MCBA antimicrobial efficacy.

Dose–response curves for L. aerotolerans when grown for 24 h in a, CA, b, LeuCA, or c, PheCA with calculated ED50 shown in red. Dose–response curves for P. anaerobius when grown for 24 h in d, CA, e, LeuCA, f, PheCA, or g, TyrCA with ED50 shown in blue. n = 4 independent cultures per strain.

Source Data

Extended Data Fig. 5 Microbiome shifts in female mice following 100 mg kg−1 MCBA gavage.

a Caecal and f faecal samples were subjected to principal coordinate analysis (PCoA) of microbiome community structure via Bray–Curtis dissimilarity after oral gavage of different MCBAs in C57BL/6 mice. Significance determined by PERMANOVA. The ratio of Firmicutes/Bacteroidota (F/B ratio) between gavage groups for b caecum and g faecal samples at day 13, with corresponding phylum-level community profiles for both c caecum and h faecal samples. Bar charts showing the top 15 bacterial groups impacting the mean decrease in accuracy of random forest classification based on MCBA gavage group for d caecal and i faecal samples from days 1−13, with higher values indicating greater contribution to the predictive model. ASVs highlighted in blue represent those that matched between caecal and faecal classifications. Comparisons between gavage groups for the top predictive ASVs in e caecal samples and j faecal samples over time, with blue graph titles indicating shared features between the top 15 predictors in both analyses. Caecum 16S analysis, n = 5 per group, 4 for PheCA; faecal 16S analysis, n = 5 per group, per timepoint. Ellipses were drawn at 95% confidence for caecum and day 13 faecal samples. Line plots show mean ± sem. In panels b, c, g and h statistical significance was determined by two-sided Wilcoxon rank-sums test against vehicle. *P < 0.05; **P < 0.01. Data in b, e and j are presented as boxplots where the middle lines are the median, lower and upper hinges represent the first and third quartiles, upper whiskers extend to maxima and lower whiskers extend to minima.

Extended Data Fig. 6 Microbiome community shifts following 10 mg kg−1 MCBA dosing via PBFM.

Timepoint-nested PERMANOVA reveals significant shifts by treatment, though significance is lost when tested within individual timepoints. n = 5 male, 5 female per treatment.

Extended Data Fig. 7 SerCA concentrations following 100 mg kg−1 feeding.

a, SerCA concentrations in murine tissue and faecal samples following 100 mg kg−1 SerCA dosing via PBFM. Data are presented as boxplots where the middle lines are the median, lower and upper hinges represent the first and third quartiles, upper whiskers extend to maxima and lower whiskers extend to minima. b, Table showing SerCA concentration by sample type, presented as mean ± sem, n = 4 mice per group.

Extended Data Fig. 8 MCBA concentrations in faecal and tissue samples following mixed MCBA dosing via PBFM.

Data are presented as the average concentration of each MCBA included in the MCBA mix (80 mg kg−1 total, 10 mg kg−1 per individual MCBA). n = 3 treatment, 2 control.

Extended Data Fig. 9 Extracted ion chromatograms of PheCA and SerCA exposed to pancreatic carboxypeptidases.

When incubated with a, 1 mM PheCA or b, 1 mM SerCA, neither pancreatic carboxypeptidase A nor pancreatic carboxypeptidase B were able to deconjugate the supplemented MCBA while still showing near-complete elimination of native substrates c, hippuryl-L-phenylalanine and d, hippuryl-L-arginine for carboxypeptidase A and carboxypeptidase B, respectively. Reactions were performed in triplicate.

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Guzior, D.V., Okros, M., Shivel, M. et al. Bile salt hydrolase acyltransferase activity expands bile acid diversity. Nature 626, 852–858 (2024). https://doi.org/10.1038/s41586-024-07017-8

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