Glycocholate

Killing assays were performed as described previously [1 (link)]. Briefly, indicated bacterial strains were grown overnight on LB agar plates with appropriate antibiotics. Prey and predator were mixed at a 10:1 or 1:1 ratios with titers normalized by OD₆₀₀ readings. Mixtures were spotted on LB agar plates with indicated supplements and incubated for 4 h at 37°C. Bacteria were harvested and serial dilutions of rifampicin-resistant prey or streptomycin-resistant predator were selectively grown on plates overnight. Sodium deoxycholate, sodium cholate, taurodeoxycholate, glycodeoxycholate, taurocholate, glycocholate, and taurine were obtained from Sigma (St. Louis MO). Glycine was obtained from Thermo Fisher Scientific (Waltham MA). Difco™ Bile Salts No.3 was obtained from BD (Mississauga ON). For assays of anaerobic bacteria, indicated bacteria were spotted on LB agar plates supplemented with 1.2 mM bile acids and incubated for 2 days under anaerobic conditions. Bacteria were scraped from the plates and plates were treated for 15 min with chloroform and incubated for an additional 15 min at 37°C [47 (link)]. This procedure ensured that all anaerobic bacteria were dead, and only their metabolites remained. Killing assays were performed on these plates.

Bovine bile (reference B3883, Sigma-Aldrich and reference 212820, Difco, BD Diagnostic Systems) was prepared at 100 mg/ml stock solution in distilled water, sterilized through 0.2 μm filters and stored at −20°C. Pure bile salts: glycocholate (GC), taurocholate (TC), glycodeoxycholate (GDC), taurodeoxycholate (TDC), glycochenodeoxycholate (GCDC), and taurochenodeoxycholate (TCDC), bile salt mix or pure deconjugated bile salts: cholate (C), deoxycholate (DC), and chenodeoxycholate (CDC), and fusidic acid were from Sigma-Aldrich and dissolved in distilled water to 12 mg/ml stock solutions, filtered through 0.2 μm pores and kept at −20°C. Choloylglycine Bile Acid Hydrolase or Bile Salt Hydrolase (BSH, EC 3.5.1.24) from Clostridium perfringens (reference C4018, Sigma-Aldrich) was prepared at 10 U/ml in distilled water and stored at −20°C. Fetal calf serum (FCS, reference A15-101, PAA Laboratories, GE Healthcare), and bovine calf serum (reference B9433, Sigma-Aldrich) were used. The NEFA-C kit used for quantitative determination of non-esterified fatty acids (NEFAs) was from Biolab, WAKO Diagnostics.

A series of bile acid standards, containing cholate (CA), chenodeoxycholate (CDCA), ursodeoxycholate (UDCA), deoxycholate (DCA), lithocholate (LCA), glycocholate (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholate (GDCA), taurocholate (TCA), taurodeoxycholate (TDCA), tauroursodeoxycholate (TUDCA), taurohyodexoycholate (THDCA), taurochenodeoxycholate (TCDCA), taurolithocholate (TLCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tauro-β-muricholate (TβMCA) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). As internal standard (IS), deoxycholic acid-2,2,4,4-d4 (DCA-d4) were obtained from CDN isotopes (Pointe-Claire, Quebec, Canada). All organic reagents for mass spectrometric analysis were HPLC grade purchased from Sigma-Aldrich (St. Louis, MO, USA). And spexin used for drug treatment was purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). M871 was purchased from R&D Systems (Minneapolis, MN, USA). SNAP37889 was purchased from Key Organics Ltd (Camelford, Cornwall, UK).