Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Apr 24.
Published in final edited form as: Methods Enzymol. 2021 Dec 31;664:85–102. doi: 10.1016/bs.mie.2021.12.002

BSH-TRAP: Bile salt hydrolase tagging and retrieval with activity-based probes

Bibudha Parasar 1, Pamela V Chang 2,3,4,*
PMCID: PMC9035283  NIHMSID: NIHMS1798451  PMID: 35331380

Abstract

Bile acids are important molecules that participate in digestion and regulate many host physiological processes, including metabolism and inflammation. Primary bile acids are biosynthesized from cholesterol in the liver, where they are conjugated to glycine and taurine before secretion into the intestines. A small fraction of these molecules remain in the gut, where they are modified by a microbial enzyme, bile salt hydrolase (BSH), which deconjugates the glycine and taurine groups. This deconjugation precedes all subsequent biotransformation in the intestines, including regioselective dehydroxylation and epimerization reactions, to produce numerous secondary bile acids. Thus, BSH is considered the gatekeeper enzyme of secondary bile acid metabolism, and, as a result, it controls the overall bile acid composition in the host. Despite the critical role that BSH plays in bile acid metabolism, there exist few tools to probe its activity in complex biological mixtures. In this chapter, we describe a chemoproteomic approach termed BSH-TRAP (bile salt hydrolase tagging and retrieval with activity-based probes) that enables visualization and identification of BSH activity in bacteria. Here, we describe application of BSH-TRAP to cultured bacterial strains and the gut microbes derived from mice. We envision that BSH-TRAP could be used to profile changes in BSH activity and identify novel BSH enzymes in complex biological samples, such as the gut microbiome.

1. Introduction

Bile acids (BAs) are a component of bile, which aids in the digestion and absorption of lipophilic nutrients in the intestines (Joyce and Gahan, 2016; Begley et al., 2005). In addition to their roles as emulsifying agents in the gut, BAs serve as signaling molecules that are recognized by specific host receptors and modulate key physiological processes (Li and Chiang, 2014). For example, BAs regulate glucose homeostasis, lipid metabolism, and energy expenditure (de Aguiar Vallim et al., 2013; Thomas et al., 2008; Tremaroli and Bäckhed, 2012). BAs also modulate immunity (Campbell et al., 2020; Hang et al., 2019), inflammation (Song et al., 2020; Devkota et al., 2012), and host defense against infection (Winkler et al., 2020; Sinha et al., 2020; Grau et al., 2020; Alavi et al., 2020; Sato et al., 2021). Thus, these metabolites represent an important class of bioactive molecules.

Primary BAs are biosynthesized de novo from cholesterol in the liver, and the solubility of the hydrophobic steroid core is increased by conjugation as an N-acyl amidate with glycine or taurine before secretion into the intestines (Ridlon et al., 2006). BAs are efficiently conserved in a process known as enterohepatic recirculation, in which the majority of these molecules are reabsorbed by the gastrointestinal tract and re-enter the liver via the portal vein. Approximately 5% of the total BA pool escapes this cycle and is extensively modified by many enzymes expressed by the gut microbiota.

Bile salt hydrolases (BSHs) are cysteine hydrolases that catalyze the first step of BA metabolism in the gut by deconjugating the glycine and taurine side chains (Begley et al., 2006). These enzymes are microbially derived and are expressed by certain intestinal bacteria (Yao et al., 2018; Jones et al., 2008). BSH is known as the gatekeeper enzyme of BA metabolism because its activity is required before all subsequent modifications by additional microbial enzymes (Foley et al., 2019). Such biotransformations may involve epimerization, dehydroxylation, and oxidation of the hydroxyl groups on the steroid core to generate a diverse pool of BAs in the gut (Funabashi et al., 2020). As a result, BSH controls the overall composition of BAs throughout the body.

Despite the importance of BSH in BA metabolism, there exist few tools for assessing its activity within the gut microbiota. Current technologies for understanding functions of microbiomes include metagenomic sequencing, which catalogues which microbes are present, transcriptomics, which profiles gene expression, and proteomics, which identifies which proteins are present (Fischbach, 2018; Koppel and Balskus, 2016). While these multi-omics approaches are powerful, they do not distinguish which proteins are enzymatically active.

Chemoproteomics has the unique ability to identify enzymatic activity within complex biological mixtures and relies on the use of activity-based probes (ABPs) to target specific enzymes (Niphakis and Cravatt, 2014). An ABP is a substrate analog that typically includes a targeting group that is specific to the enzyme of interest, a covalent warhead that reacts selectively with an active-site nucleophile, and a click chemistry handle that can undergo bioorthogonal chemistries to label the active enzyme with an imaging agent or an affinity probe (Scinto et al., 2021). ABPs have been applied to detect enzymatic activity within complex biological samples using either visualization with imaging modalities or protein identification by pull-down using affinity-based reagents, followed by mass spectrometry (MS)-based proteomics.

We have developed chemoproteomic approaches for identifying which BSH enzymes are active within model gut anaerobes and the gut microbiota (Figure 1) (Parasar et al., 2019). Our first-generation ABP included an acyloxymethylketone covalent warhead (Kato et al., 2005), a mild electrophile that is highly selective for reactive cysteine residues (Ch-AOMK, Figure 2). Others have deployed this general strategy to develop covalent BSH inhibitors (Adhikari et al., 2021, 2020) and ABPs for BSH imaging that rely on fluorescence (Sveistyte et al., 2020; Brandvold et al., 2019) and bioluminescence (Khodakivskyi et al., 2021). Additional BSH ABPs have recently been reported with alternative covalent warheads that target the active-site cysteine residue (Brandvold et al., 2021). We have termed our chemoproteomic approach for identifying BSH activity in the gut microbiota BSH-TRAP (Bile salt hydrolase tagging and retrieval with activity-based probes). Here, we describe the application of BSH-TRAP to identify BSH activity within cultured bacterial strains and the gut microbiota from mice.

Fig. 1.

Fig. 1.

Open in a new tab

Chemoproteomic, activity-based approach for profiling bile salt hydrolase (BSH) activity within cultured bacterial strains and the gut microbiome.

Fig. 2.

Fig. 2.

Open in a new tab

Synthesis of Ch-AOMK.

2. General methods

2.1. Equipment

NMR spectra were obtained using a Bruker INOVA 500 NMR spectrometer. Reverse phase HPLC was performed using a Shimadzu system equipped with a CBM-20A controller, SPD20AV UV-Vis detector, LC-20AR liquid chromatograph unit, FRC-10A fraction collector, and an Epic Polar 5μm 120Å C18 analytical column (4.6 × 250 mm) at a flow rate of 1 mL/min or a semipreparative column (10 × 250 mm) at a flow rate of 4 mL/min. Anaerobic chamber (Coy Lab, model AC16–113) was maintained with the gas composition (3% hydrogen, 20% carbon dioxide, and 77% nitrogen). A Coy Lab (model 2000) incubator was used inside the anaerobic chamber for the growth of anaerobic bacteria. UV absorbance readings were measured on a Bio-Tek PowerWave XS microplate spectrophotometer. Bacterial lysis was performed using a BioSpec MiniBeadBeater-16, Model 607.

2.2. Materials

All chemical reagents were of analytical grade, obtained from commercial suppliers, and used without further purification unless otherwise noted. Organic extracts were dried over Na2SO4, and solvents were removed with a rotary evaporator at reduced pressure (20 torr), unless otherwise noted. Flash chromatography was performed using Silicycle Siliaflash P60 40–63Å 230–400 mesh silica gel. Analytical thin layer chromatography (TLC) was performed on glass-backed TLC 60 Å silica gel plates, and compounds were visualized by staining with ceric ammonium molybdate and the absorbance of UV light (λ = 254 nm or 365 nm). HPLC samples were filtered with a MillexLH syringe filter equipped with a 0.45 μm PTFE membrane prior to injection. Bifidobacterium longum subsp. infantis ATCC15697 and Bifidobacterium bifidum DSM20456 strains were purchased from the American Type Culture Collection. Bifidobacterium species were grown in Bifidobacterium broth supplied by HIMedia (M1395). Nycodenz was obtained from VWR (#100334–594). The DC protein assay was obtained from Bio-Rad. Zirconia/silica beads (0.1 mm diameter) were obtained from BioSpec Products. High capacity streptavidin agarose beads were purchased from Thermo Fisher Scientific. Rhodamine110-based Fluor 488-alkyne was obtained from Sigma-Aldrich, and biotin-PEG4-alkyne (biotin-alkyne) was obtained from Click Chemistry Tools. Streptavidin-conjugated horse radish peroxidase (streptavidin-HRP) was obtained from Genetex (GTX85912). Glass beads (3 mm) were obtained from Fisher Scientific (11–312A). SYPRO Ruby Protein Gel Stain was obtained from Thermo Fisher Scientific. Costar 0.22 μm spin filter was obtained from Corning (#8161). Sequencing Grade Modified Trypsin was obtained from Promega.

3. Synthesis of bile salt hydrolase (BSH) activity-based probe

Azidocholic acid (Thota et al., 2014) (300 mg, 0.7 mmol) was dissolved in anhydrous THF (3.64 mL) and stirred in a dry ice/acetone bath at −15 °C for 5 min. N-methylmorpholine (98.1 μL, 0.866 mmol, 1.25 eq.) and isobutyl chloroformate (104 μL, 0.8 mmol, 1.15 eq.) were sequentially added to this solution, and the mixture was stirred at −15 °C for an additional 30 min, during which a white precipitate formed. The reaction was brought to 0 °C. Ethereal diazomethane was generated in situ according to the procedure reported in the Sigma-Aldrich technical bulletin (AL-180). A flame polished glass pipette was used to add diazomethane (3 mmol, 3.75 eq.) dropwise to the reaction mixture at 0 °C, and the reaction was slowly warmed to room temperature over 4 h. To generate the corresponding bromomethyl ketone, the reaction mixture was cooled to 0 °C. Hydrogen bromide (33 w% in acetic acid, 5 mL, 75 mmol, 107 eq.) was mixed with 10 mL of water and added dropwise to the reaction mixture until the evolution of nitrogen gas stopped. The mixture was diluted with ethyl acetate and transferred to a separatory funnel. The organic layer was washed sequentially with water, brine, and NaHCO3, then dried over anhydrous Na2SO4. The organic layers were combined and rotovapped to yield a sticky yellow solid.

The crude was dissolved in dry DMF (0.7 mL) and was stirred at room temperature under nitrogen, and 2,6-dimethylbenzoic acid (30 mg, 0.2 mmol, 0.28 eq.) and sodium bicarbonate (17 mg, 0.2 mmol, 0.28 eq.) were added. After 2 h, the reaction mixture was diluted with ethyl acetate and transferred to a separatory funnel. The organic layer was washed with ddH2O three times and dried over anhydrous Na2SO4. The crude was purified by flash column chromatography (2% MeOH in DCM) to yield a sticky solid. The solid was dissolved in DMSO and further purified by HPLC (HPLC grade water:acetonitrile gradient varied from 100:0 to 55:45 over 50 min with a curve value of −4) to generate the purified compound as a white powder (Ch-AOMK, 41% yield). TLC (CH2Cl2:MeOH, 99:1 v/v): Rf = 0.6; 1H NMR (500 MHz, DMSO-d6): δ 7.27 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 3.75 Hz, 2H), 5.05 (s, 2H), 4.17 (m, 2H), 3.80 (s, 1H), 3.63 (s, 1H), 3.23 (m, 1H), 2.41 (m, 2H), 2.32 (s, 6H), 2.10 (m, 2H), 1.99 (s, 1H), 1.83 (m, 2H), 1.75 (m, 2H), 1.66 (m, 2H), 1.58 (m, 2H), 1.39 (m, 7H), 1.23 (m, 2H), 1.00 (m, 1H), 0.95 (d, J = 2.5 Hz, 4H), 0.85 (s, 3H), 0.6 (s, 3H); 13C NMR (500 MHz, DMSO-d6): 03B4 204.58, 177.34, 168.75, 135.19, 133.49, 130.03, 128.01, 71.45, 68.79, 66.57, 61.25, 46.51, 46.24, 41.97, 41.86, 35.51, 35.46, 35.37, 34.97, 34.79, 29.38, 28.92, 27.68, 26.79, 26.60, 23.21, 22.95, 19.77, 17.60, 12.80; ESI-MS (m/z): [M-H]− calcd. For C34H48N3O5−, 578.3599; found, 578.3570.

Safety statement: Significant hazards were mitigated in the generation of diazomethane by using smoothened glass joints, a blast shield, and loosely sealing the reaction vessel. Afterwards, the syringes, needles, and glassware were quenched with acetic acid.

4. Application of BSH-TRAP to cultured bacterial strains

4.1. General methods

4.1.1 The day before bacterial (e.g., B. longum and B. bifidum) cultures were started, media was prepared according to the manufacturer’s recommendation and allowed to equilibrate with the anaerobic gas mix (5% H2, 20% CO2, 75% N2) for 24 h in the anaerobic chamber. The next day, 5 mL of the media was inoculated with bacteria. The bacterial culture was incubated at 37 °C until it reached stationary phase (OD600 = 1.2–1.5) and diluted into 50 mL of media. After reaching stationary phase, the bacterial culture was centrifuged at 4,500 x g for 15 min at 4 °C to pellet the bacteria. The supernatant was discarded, and the bacterial pellet was flash-frozen and stored at - 80 °C until further use.

4.1.2 The bacterial pellet was thawed on ice and washed three times with 5 mL of cold 1X phosphate buffered saline (PBS) to remove any residual media. (The samples were kept on ice in the subsequent steps except during bacterial lysis.) The bacteria were aliquoted into several microcentrifuge tubes, each containing ~80–100 μL of the bacteria pellet for subsequent lysis. The bacteria were resuspended in 200 μL lysis buffer (20 mM sodium phosphate buffer, pH 7.4, 1 mM EDTA, 10% glycerol), and the volume was adjusted with lysis buffer to a final volume of 300 μL. To the bacterial suspension was added 3 μL of 1M dithiothreitol (DTT in ddH2O) and 3 μL of 1M phenylmethylsulfonylfluoride (PMSF in ddH2O). The bacterial suspension was transferred to a 2 mL screw-top tube containing 330 mg of zirconia/silica beads (0.1 mm diameter), and the samples were rested on ice for 3 min. The tubes were then subjected to 6 cycles of bead beating (3400 rpm; 1 cycle: 1 min beating, 3 min rest on ice). After lysis, the tubes were centrifuged at 17,000 x g for 15 min at 4 °C. The supernatant was transferred to a clean microcentrifuge tube and quantified using the DC protein assay.

4.2. Fluorescence gel in situ BSH activity assay

4.2.1 To set up a fluorescence assay (Figure 3), lysates (100 μg) were treated with vehicle (DMSO), Ch-AOMK (500 μM), iodoacetamide (20 mM), and a combination of Ch-AOMK (500 μM) and iodoacetamide (20 mM) in 10X reaction buffer (500 mM sodium acetate, pH 5.5). The reactions (total volume 20 μL) were incubated at 37 °C for 24 h.

Fig. 3.

Fig. 3.

Open in a new tab

Schematic diagram of identifying BSH activity by fluorescence-based in gel activity assay and mass spectrometry (MS)-based proteomic analysis, following click chemistry.

4.2.2 The samples were quenched by adding cold methanol (200 μL), chloroform (75 μL), and ddH2O (150 μL). The samples were vortexed briefly and centrifuged at 17,000 x g for 20 min at 4 °C after which the mixture forms an organic layer and an aqueous layer. Proteins are precipitated at the interphase as a disc, and the top aqueous layer was discarded.

4.2.3 One milliliter of cold methanol was added to the bottom organic layer, and the samples were inverted a few times. The tubes were centrifuged at 17,000 x g for 10 min at 4 °C. The supernatant was decanted, and the precipitate was washed again with cold methanol (1 mL) by inverting the samples a few times. The samples were again centrifuged at 17,000 x g for 10 min at 4 °C, decanted, and the residual methanol was carefully removed with a pipette. The precipitate was air-dried at room temperature for 15 min. After the precipitate was dry, 16 μL of click buffer (0.1M sodium phosphate buffer, pH 7.4, 4% sodium dodecyl sulfate, SDS) was added, and the samples were incubated at 37 °C until the proteins were fully solubilized.

4.2.4 During this period, the click chemistry reagents were prepared in which the copper sulfate and ascorbate solution were prepared fresh each time. For each click chemistry reaction, 20 μL THPTA (20 mM) and 2.5 μL CuSO4·5H2O (80 mM) was pre-mixed to form the in-situ copper complex. After the protein precipitate is fully solubilized in the click buffer, the following reagents were added sequentially: 1 μL of fluorophore-alkyne (Fluor-488 alkyne, 1 mM in DMSO), 2.25 μL copper-THPTA complex mix in dd2HO, and 1 μL of sodium ascorbate (1 M in ddH2O). The samples (20 μL) were mixed by vortexing between each addition and incubated in the dark at 37 °C for 2 h.

4.2.5 After the click chemistry reaction, cold methanol (200 μL), chloroform (75 μL), and ddH2O (150 μL) was added to each sample. The samples were vortexed briefly and centrifuged at 17,000 x g for 20 min at 4 °C. The top aqueous layer was discarded, and cold methanol (1 mL) was added to the bottom organic layer, which was then inverted a few times. The samples were centrifuged at 17,000 x g for 10 min at 4 °C, decanted, and the precipitate was washed with cold methanol (1 mL) by inverting the sampls a few times. The samples were again centrifuged at 17,000 x g for 10 min at 4 °C and decanted. The residual methanol was carefully removed with a pipette, and the precipitate was air-dried at room temperature for 15 min. Afterwards, 30 μL of 1X Laemmli buffer was added, and the samples were boiled at 95 °C for 20 min until the precipitate was fully dissolved.

4.2.6 The samples were resolved by SDS-PAGE (12%), imaged by fluorescence gel scanning, and stained with Coomassie Brilliant Blue (R-250, 1% (w/v) in 30% methanol, 10% acetic acid) for 10–15 min and subsequently destained in destaining solution (30% methanol, 5% acetic acid).

4.3. Immunoprecipitation of BSHs followed by streptavidin blot analysis

4.3.1 For the immunoprecipitation (Figure 3), lysates (2.5 mg) were treated with vehicle (DMSO) or Ch-AOMK (500 μM) in 10X reaction buffer (500 mM sodium acetate, pH 5.5). The samples (total volume 1 mL) were incubated at 37 °C for 24 h. The samples were quenched by adding cold methanol (400 μL), chloroform (150 μL), and ddH2O (300 μL), followed by precipitation as in 4.2.2.

4.3.2 One milliliter of cold methanol was added, and the samples were inverted a few times. The samples were centrifuged at 17,000 x g for 10 min at 4 °C. The supernatant was decanted, and the precipitate was washed again with cold methanol (1 mL) by inverting the samples a few times. The samples were sonicated and again centrifuged at 17,000 x g for 10 min at 4 °C, then decanted, and the residual methanol was carefully removed by a pipette. The precipitate was air-dried at room temperature for 15 min, after which 170 μL of click buffer (0.1M sodium phosphate buffer, pH 7.4, 4% SDS) was added, and the samples were sonicated until the protein was fully solubilized.

4.3.3 Click chemistry reagents were prepared and a 200 μL reaction was set up as in 4.2.4, using biotin-alkyne (100 μM in DMSO).

4.3.4 After the click chemistry reaction, cold methanol (200 μL), chloroform (75 μL), and ddH2O (150 μL) was added to the bottom organic layer. The samples were vortexed briefly and centrifuged at 17,000 x g for 20 min at 4 °C. The top aqueous layer was discarded, and cold methanol (1 mL) was added to the bottom organic layer, which was then inverted a few times. The samples were centrifuged at 17,000 x g for 10 min at 4 °C. The samples were decanted, and the precipitate was washed with cold methanol (1 mL) by inverting the samples a few times. The samples were sonicated, centrifuged at 17,000 x g for 10 min at 4 °C, and decanted. The residual methanol was carefully removed by a pipette, and the precipitate was air-dried at room temperature or 37 °C.

4.3.5 Afterwards, 166 μL of 1.2% (w/v) SDS in 1X PBS was added, and the samples were sonicated. The samples were then diluted by adding 833 μL of 1X PBS, bringing the final volume to 1 mL (0.2% (w/v) SDS). The input samples (10 μL) were collected.

4.3.6 In the meantime, 20 μL of streptavidin-agarose beads were rinsed with 0.2% SDS in 1X PBS (3 × 500 μL). The samples were transferred to the tube containing the beads and rotated for 1 h at room temperature.

4.3.7 Next, the samples were spun down, and the supernatant was saved as “flow through.” The beads were sequentially rinsed with 1%, 0.5% and 0.2% SDS in 1X PBS, and the supernatant was carefully removed after the last wash. The beads were resuspended in 30 μL of 2X Laemmli buffer and boiled at 95 °C for 20 min. The samples were centrifuged, and the supernatant (e.g., “elution”) was collected. The samples were resolved by SDS-PAGE (12%), followed by streptavidin blotting with streptavidin-HRP and silver staining.

4.4. Immunoprecipitation of BSHs followed by mass spectrometry (MS)-based proteomics

4.4.1 The steps for sample preparation for shotgun proteomics are very similar to the steps for

4.3. Briefly, lysates (10 mg) were treated as in 4.3.1–4.3.6 at the appropriate scale.

4.4.2 General protocol for in-gel digestion of proteins: To elute the proteins, 120 μL of 2X SDS was added to the beads, and the samples were boiled at 95 °C for 30 min. The tubes were centrifuged at 13,000 x g for 1 min at room temperature, and the supernatant was collected. The eluted protein lysates (100 μL) resolved by SDS-PAGE (12%). Then, the gel was fixed in the fixation buffer (50% methanol, 10% acetic acid, 40% water) for at least 1 h and transferred to gel storage buffer (10% methanol, 7% acetic acid, 83% water) before excision. (Simultaneously, a streptavidin blot and silver staining was carried out with 20 μL of the samples as quality control for proteomics.) The proteins were visualized with SYPRO Ruby Protein Gel Stain, and the desired bands were cut into ~1 mm cubes and subjected to in-gel digestion, followed by extraction of the tryptic peptide as reported previously (Yang et al., 2007). The excised gel pieces were washed consecutively in 200 μL of ddH2O, 100 mM ammonium bicarbonate/acetonitrile (1:1), and acetonitrile. The gel pieces were subsequently reduced with 250 μL of 10 mM DTT in 100 mM ammonium bicarbonate for 1 h at 56 °C and alkylated with 260 μL of 55 mM iodoacetamide in 100 mM ammonium bicarbonate at room temperature in the dark for 1 h. After washing, the gel slices were dried and rehydrated with 10 μL of trypsin reconstituted in 50 mM ammonium bicarbonate, 10% acetonitrile at 37 °C for 16 h. The digested peptides were extracted twice with 20 μL of 50% acetonitrile, 5% formic acid and once with 20 μL of 90% acetonitrile, 5% formic acid. Extracts from each sample were combined and the extracted peptide solution was filtered with a Costar 0.22 μm spin filter and lyophilized.

4.4.3 On-bead digestion for identification of proteins: After washing the beads with decreasing concentrations of SDS in 1X PBS (1%, 0.5% and 0.2%), the beads were incubated with 500 μL of 6 M urea in 1X PBS supplemented with 10 mM tris(2-carboxyethyl)phosphine at 37 °C for 30 min, after which 25 μL of 400 mM iodoacetamide was added. The samples were further incubated at 37 °C for 30 min. In the meantime, trypsin was activated by heating at 30 °C for 15 min. The beads were washed with 1 mL of 1M urea in 1X PBS, after which the samples were incubated with 2 μg trypsin (in 200 μL of 1M urea with 1 mM CaCl2 in 1X PBS, pH 8.0) at 37 °C overnight with gentle rotation. The supernatant was removed the following day, and 800 μL of ddH2O was added to bring the final volume to 1 mL. The samples were acidified with 1N HCl (6 μL) to reach a pH of ~2.6 to inactivate trypsin and lyophilized.

4.4.4 The samples were analyzed by shotgun proteomics as previously reported (Parasar et al., 2019).

5. Application of BSH-TRAP to mouse gut microbiome samples

5.1. General procedures

5.1.1 Mouse fecal pellets were collected on dry ice. To 2.5 g of fecal pellets was added 10 mL of cold 1X PBS, and the pellets were crushed with a mortar and pestle. The samples were transferred to a 50 mL tube containing 5–6 large glass beads (3 mm) and vortexed to break down larger particles. The sample was centrifuge at 300 x g for 5 min at 4 °C, and the supernatant was transferred to a 50 mL tube. A second round of bacteria extraction was performed by adding 3 mL of 1X PBS. The combined supernatants were centrifuged at 300 x g for 5 min at 4 °C to remove any remaining particles. A density gradient was prepared by adding 0.5 mL of Nycodenz (50%) to several 1.5 mL microcentrifuge tubes. One milliliter of the bacteria slurry was carefully added to the sample in a manner that does not disturb the Nycodenz gradient.

5.1.2 The samples were centrifuged at 10,000 x g for 40 min at 4 °C. At this point, i) larger debris sank to the bottom, ii) bacteria were concentrated at the interface, and iii) small molecules and other components remained in the top layer. The top layer was carefully discarded, and the bacteria were transferred to a 15 mL tube. The bacteria were washed by resuspension with double the volume of 1X PBS and aliquoted into 5 microcentrifuge tubes. The samples were centrifuged at 17,000 x g for 10 min at 4 °C, and each tube was washed again by resuspension with 1 mL 1X PBS and centrifugation (17,000 x g for 10 min at 4 °C) to completely remove the Nycodenz.

5.2. Fluorescence assay, immunoprecipitation, and MS-based proteomics of mouse microbiome samples

5.2.1 The fecal bacteria pellet was lysed as in 4.2.1, and the fluorescence assay was carried out as in 4.2. For the immunoprecipitation, samples were treated as in 4.3.1, with the following modifications:

5.2.2 Labeling of bacterial lysates with Ch-AOMK (100 μM) was carried out at 37 °C for 12 h, followed by quenching with trichloroacetic acid (TCA, 15% w/v). The samples were incubated on ice for 30 min, centrifuged at 17,000 x g for 5 min at 4 °C, and decanted. The precipitate was washed twice with cold acetone (1 mL) with sonication, followed by centrifugation at 17,000 x g for 5 min at 4 °C. Residual acetone was carefully removed by a pipette, and the precipitate was air-dried. Afterwards, 170 μL of click buffer (0.1 M sodium phosphate buffer, pH 7.4, 4% SDS) was added, and the samples were sonicated to dissolve the precipitate.

5.2.3 Click chemistry reagents were prepared as in 4.3.3. After the protein precipitate was fully solubilized in the click buffer, the following reagents were added sequentially: 2 μL of biotin- alkyne, 22.5 μL copper-THPTA complex mix, and 10 μL of sodium ascorbate (200 μL final volume). The samples were mixed by vortexing between each addition and incubated in the dark at 37 °C for 2 h.

5.2.4 After the click chemistry reaction, the samples were precipitated by adding TCA (15% w/v) as in 5.2.2. The samples were then immunoprecipitated as in 4.3.5–4.3.7. Alternatively, lysate (10 mg) was immunoprecipitated as in 4.4 for shotgun proteomic analyses (Parasar et al., 2019). Metagenomic sequencing was used to identify the bacteria present in the fecal samples.

6. General considerations for BSH-TRAP

6.1 Fluorescence assay: If the protein precipitate is small, proteins can be recovered from the side of the tubes by scraping with a pipet tip. In this case, it is better to air-dry the protein precipitate at room temperature or 37 °C to avoid loss. A close watch needs to be kept on the sample while drying the precipitate. As the precipitate dries, the color changes from white to off-white, and overdrying can lead to further changes in color to yellow, brown, and eventually black. Caution: Overdrying can prevent the protein precipitate from going back into solution.

6.2 Mouse fecal collection: Care must be taken not to mix the fecal pellets with mouse urine or leave the pellets in the cage for too long to prevent exposure of gut anaerobes to oxygen. Pellets can be flash frozen and stored at −80 °C until further use.

Acknowledgments

We thank members of the Chang Lab for their assistance with preparing this chapter. Research in the Chang Lab is supported by a Beckman Young Investigator Award from the Arnold and Mabel Beckman Foundation and the National Institutes of Health (NIH R35GM133501).

References

  1. Adhikari AA, Ramachandran D, Chaudhari SN, Powell CE, Li W, McCurry MD, Banks AS, and Devlin AS 2021. A Gut-Restricted Lithocholic Acid Analog as an Inhibitor of Gut Bacterial Bile Salt Hydrolases. ACS Chem. Biol doi: 10.1021/acschembio.1c00192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adhikari AA, Seegar TCM, Ficarro SB, McCurry MD, Ramachandran D, Yao L, Chaudhari SN, Ndousse-Fetter S, Banks AS, Marto JA, Blacklow SC, and Devlin AS 2020. Development of a covalent inhibitor of gut bacterial bile salt hydrolases. Nat. Chem. Biol 2020. 16:1–9. doi: 10.1038/s41589-020-0467-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. de Aguiar Vallim TQ, Tarling EJ, and Edwards PA 2013. Pleiotropic Roles of Bile Acids in Metabolism. Cell Metab. 17:657–669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alavi S, Mitchell JD, Cho JY, Liu R, Macbeth JC, and Hsiao A. 2020. Interpersonal Gut Microbiome Variation Drives Susceptibility and Resistance to Cholera Infection. Cell. 181:1533–1546.e13. doi: 10.1016/j.cell.2020.05.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Begley M, Hill C, and Gahan CGM 2006. Bile Salt Hydrolase Activity in Probiotics. Appl. Environ. Microbiol 72:1729–1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Begley M.Ã. ire, Gahan CGM, and Hill C. 2005. The interaction between bacteria and bile. FEMS Microbiol. Rev 29:625–651. [DOI] [PubMed] [Google Scholar]
  7. Brandvold KR, Miller CJ, Volk RF, Killinger BJ, Whidbey C, and Wright AT 2021. Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes. ChemBioChem. 1–9. doi: 10.1002/cbic.202000748. [DOI] [PubMed] [Google Scholar]
  8. Brandvold KR, Weaver JM, Whidbey C, and Wright AT 2019. A continuous fluorescence assay for simple quantification of bile salt hydrolase activity in the gut microbiome. Sci. Rep 9:1359. doi: 10.1038/s41598-018-37656-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Campbell C, McKenney PT, Konstantinovsky D, Isaeva OI, Schizas M, Verter J, Mai C, Jin WB, Guo CJ, Violante S, Ramos RJ, Cross JR, Kadaveru K, Hambor J, and Rudensky AY 2020. Bacterial metabolism of bile acids promotes generation of peripheral regulatory T cells. Nature. 581:475–479. doi: 10.1038/s41586-020-2193-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Devkota S, Wang Y, Musch MW, Leone V, Fehlner-Peach H, Nadimpalli A, Antonopoulos DA, Jabri B, and Chang EB 2012. Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in Il10−/− mice. Nature. 487:104–108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. doi. doi: 10.1038/nature11225. [DOI] [Google Scholar]
  12. Fischbach MA 2018. Microbiome: Focus on Causation and Mechanism. Cell. 174:785–790. doi: 10.1016/j.cell.2018.07.038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foley MH, Flaherty SO, Barrangou R, and Theriot CM 2019. Bile salt hydrolases : Gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract. PLoS Pathog. 1–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Funabashi M, Grove TL, Wang M, Varma Y, McFadden ME, Brown LC, Guo C, Higginbottom S, Almo SC, and Fischbach MA 2020. A metabolic pathway for bile acid dehydroxylation by the gut microbiome. Nature. 582:566–570. doi: 10.1038/s41586-0202396-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grau KR, Zhu S, Peterson ST, Helm EW, Philip D, Phillips M, Hernandez A, Turula H, Frasse P, Graziano VR, Wilen CB, Wobus CE, Baldridge MT, and Karst SM 2020. The intestinal regionalization of acute norovirus infection is regulated by the microbiota via bile acid-mediated priming of type III interferon. Nat. Microbiol 5:84–92. doi: 10.1038/s41564019-0602-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hang S, Paik D, Yao L, Kim E, Jamma T, Lu J, Ha S, Nelson BN, Kelly SP, Wu L, Zheng Y, Longman RS, Rastinejad F, Devlin AS, Krout MR, Fischbach MA, Littman DR, and Huh JR 2019. Bile acid metabolites control Th17 and Treg cell differentiation. Nature. 576:143–148. doi: 10.1038/s41586-019-1785-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones BV, Begley M, Hill C, Gahan CGM, and Marchesi JR 2008. Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome. Proc. Natl. Acad. Sci. U.S.A 105:13580–13585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Joyce SA, and Gahan CGM 2016. Bile Acid Modifications at the Microbe-Host Interface: Potential for Nutraceutical and Pharmaceutical Interventions in Host Health. Annu. Rev. Food Sci. Technol 7:313–333. [DOI] [PubMed] [Google Scholar]
  19. Kato D, Boatright KM, Berger AB, Nazif T, Blum G, Ryan C, Chehade KAH, Salvesen GS, and Bogyo M. 2005. Activity-based probes that target diverse cysteine protease families. Nat. Chem. Biol 1:33–38. [DOI] [PubMed] [Google Scholar]
  20. Khodakivskyi PV, Lauber CL, Yevtodiyenko A, Bazhin AA, Bruce S, Ringel-kulka T, Ringel Y, Bétrisey B, Torres J, Hu J, Chou CJ, and Goun EA 2021. Noninvasive imaging and quantification of bile salt hydrolase activity : From bacteria to humans. Sci. Adv 7:eaaz9857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Koppel N, and Balskus EP 2016. Exploring and Understanding the Biochemical Diversity of the Human Microbiota. Cell Chem. Biol 23:18–30. [DOI] [PubMed] [Google Scholar]
  22. Li T, and Chiang JYL 2014. Bile Acid Signaling in Metabolic Disease and Drug Therapy. Pharmacol. Rev 66:948–983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Niphakis MJ, and Cravatt BF 2014. Enzyme Inhibitor Discovery by Activity-Based Protein Profiling. Annu. Rev. Biochem 83:341–377. [DOI] [PubMed] [Google Scholar]
  24. Parasar B, Zhou H, Xiao X, Shi Q, Brito IL, and Chang PV 2019. Chemoproteomic Profiling of Gut Microbiota-Associated Bile Salt Hydrolase Activity. ACS Cent. Sci 5:867–873. doi: 10.1021/acscentsci.9b00147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ridlon JM, Kang D-J, and Hylemon PB 2006. Bile salt biotransformations by human intestinal bacteria. J. Lipid Res 47:241–259. [DOI] [PubMed] [Google Scholar]
  26. Sato Y, Atarashi K, Plichta DR, Arai Y, Sasajima S, Kearney SM, Suda W, Takeshita K, Sasaki T, Okamoto S, Skelly AN, Okamura Y, Vlamakis H, Li Y, Tanoue T, Takei H, Nittono H, Narushima S, Irie J, Itoh H, Moriya K, Sugiura Y, Suematsu M, Moritoki N, Shibata S, Littman DR, Fischbach MA, Uwamino Y, Inoue T, Honda A, Hattori M, Murai T, Xavier RJ, Hirose N, and Honda K. 2021. Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians. Nature. doi: 10.1038/s41586-021-03832-5. [DOI] [PubMed] [Google Scholar]
  27. Scinto SL, Bilodeau DA, Hincapie R, Lee W, Nguyen SS, Xu M, am Ende CW, Finn MG, Lang K, Lin Q, Pezacki JP, Prescher JA, Robillard MS, and Fox JM 2021. Bioorthogonal chemistry. Nat. Rev. Methods Prim 1:30. doi: 10.1038/s43586-021-00028-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sinha SR, Haileselassie Y, Nguyen LP, Tropini C, Wang M, Becker LS, Sim D, Jarr K, Spear ET, Singh G, Namkoong H, Bittinger K, Fischbach MA, Sonnenburg JL, and Habtezion A. 2020. Dysbiosis-Induced Secondary Bile Acid Deficiency Promotes Intestinal Inflammation. Cell Host Microbe. 1–12. doi: 10.1016/j.chom.2020.01.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Song X, Sun X, Oh SF, Wu M, Zhang Y, Zheng W, Geva-Zatorsky N, Jupp R, Mathis D, Benoist C, and Kasper DL 2020. Microbial bile acid metabolites modulate gut RORγ+ regulatory T cell homeostasis. Nature. 577:410–415. doi: 10.1038/s41586-019-1865-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sveistyte A, Gibbins T, Tyrrell KJ, Miller CJ, Foley MH, Plymale AE, Wright AT, and Brandvold KR 2020. Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates. ChemBioChem. 21:3539–3543. doi: 10.1002/cbic.202000370. [DOI] [PubMed] [Google Scholar]
  31. Thomas C, Pellicciari R, Pruzanski M, Auwerx J, and Schoonjans K. 2008. Targeting bile-acid signalling for metabolic diseases. Nat. Rev. Drug Discov 7:678–693. [DOI] [PubMed] [Google Scholar]
  32. Thota BNS, Savyasachi AJ, Lukashev N, Beletskaya I, and Maitra U. 2014. Tripodal Bile Acid Architectures Based on a Triarylphosphine Oxide Core Obtained by Copper-Catalysed [1,3]-Dipolar Cycloaddition: Synthesis and Preliminary Aggregation Studies. European J. Org. Chem. 2014:1406–1415. [Google Scholar]
  33. Tremaroli V, and Bäckhed F. 2012. Functional interactions between the gut microbiota and host metabolism. Nature. 489:242–249. [DOI] [PubMed] [Google Scholar]
  34. Winkler ES, Shrihari S, Hykes BL, Handley SA, Andhey PS, Huang Y-JS, Swain A, Droit L, Chebrolu KK, Mack M, Vanlandingham DL, Thackray LB, Cella M, Colonna M, Artyomov MN, Stappenbeck TS, and Diamond MS 2020. The Intestinal Microbiome Restricts Alphavirus Infection and Dissemination through a Bile Acid-Type I IFN Signaling Axis. Cell. 1–18. doi: 10.1016/j.cell.2020.06.029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yang Y, Thannhausser TW, Li L, and Zhang S. 2007. Development of an integrated approach for evaluation of 2-D gel image analysis: Impact of multiple proteins in single spots on comparative proteomics in conventional 2-D gel/MALDI workflow. Electrophoresis. 28:2080–2084. doi: 10.1002/elps.200600524. [DOI] [PubMed] [Google Scholar]
  36. Yao L., Seaton SC, Ndousse-Fetter S, Adhikari AA, DiBenedetto N, Mina AI, Banks AS, Bry L, and Devlin AS 2018. A selective gut bacterial bile salt hydrolase alters host metabolism. Elife. 7:e37182. doi: 10.7554/eLife.37182. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES