Cholate

Human M₂ muscarinic receptor bearing the c-Myc or FLAG epitope at the N-terminus was expressed in Sf9 cells as described previously (Redka et al., 2014 (link); Shivnaraine et al., 2012 (link)) and in Appendix 1. The cells were harvested and solubilized in digitonin–cholate (0.86% digitonin, Wako Chemicals USA; 0.17% cholate, Sigma-Aldrich), and aliquots of the extract were removed for electrophoresis and binding assays. The receptor was purified in predominantly monomeric form by successive passage on DEAE-Sepharose, ABT-Sepharose, and hydroxyapatite. The final concentrations of digitonin and cholate were 0.1% and 0.02%, respectively. Purified receptor was stored at −75°C. Further details regarding the purification and the nature of the purified receptor have been described previously (Redka et al., 2014 (link)).

Caffeine, cholesterol, sucrose, and cholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). CCl₄ was obtained from J.T. Backer (Xalostoc, Mexico City, Mexico). Gloria^® brand unsalted butter (Cremería Americana S.A. de C.V., Mexico City, Mexico) and rennet casein powder 30 mesh size (Irish Dairy Board Proteins and Ingredients, Dublin, Ireland) were used in this study.

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).

POPC and POPS lipids were purchased from Avanti Polar Lipids in chloroform with concentrations determined by total phosphate analysis. Nanodiscs containing 100% POPC and 70% POPC/30% POPS were prepared based on a published protocol with minor modifications^{76 (link)}. Briefly, stock lipids were mixed together at room temperature, slowly dried using argon gas while incubating in a bead heat bath at 37 °C, and then put on a vacuum lyophilyzer overnight to remove all residual organic solvents. The dried lipids were reconstituted to 65 mM with 130 mM cholate (Sigma -Aldrich) in 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.5 mM EDTA buffer, and mixed with MSP ΔH5 in a 30:1 lipid to protein ratio. The mixtures were mixed on a Nutator at 4 °C for 1 hour, and then 0.6 g/mL of washed bio-beads (Bio-Rad laboratory) were added and incubated at 4 °C for additional 4.5 hours. Afterwards, the nanodiscs were removed from the bio-beads by careful pipetting and purified using gel filtration chromatography on an AKTA FPLC with a GE Superdex 200 increase column (10 × 300 mm) with the mobile buffer containing 20 mM HEPES pH 7.4, 100 mM NaCl, and 0.5 mM EDTA at 0.5 mL/min flow rate. Peak fractions were pooled and analyzed by dynamic light scattering to assess homogeneity; pooled fractions consistently had a polydispersity of less than 15% and diameter of ~7.2–7.5 Å.

Heat, oxidative, bile salt and acid challenges were applied to the cultures at the start of stationary phase (when the maximal OD was reached after 48 h of growth) as described previously (Leverrier et al., 2004 (link); Gaucher et al., 2019a (link)). The level of each challenge was fixed in order to trigger cell death in naïve propionibacteria but not in adapted ones. The heat challenge was performed by placing 2 mL (in a 15 mL polystyrene Falcon^TM tube) of P. freudenreichii culture in a water bath at 60°C for 10 min (Leverrier et al., 2004 (link)). The oxidative challenge was performed by adding 1.25 mM hydrogen peroxide (Labogros, France) to 2 mL P. freudenreichii culture for 1 h at 30°C (Serata et al., 2016 (link); Gaucher et al., 2019a (link)). For the acid challenge, cultures were centrifuged (6,000 × g, 10 min) and re-suspended in the same growth medium, but adjusted to pH 2.0 using HCl, prior to 1 h incubation at 30°C (Jan et al., 2001 (link)). The bile salts challenge was performed by adding 1 g.L^–1 of a bile salts mixture (an equimolar mixture of cholate and deoxycholate; Sigma Chemical, St. Louis, MO, United States) to the culture before incubation for 1 h at 30°C (Leverrier et al., 2003 (link)). CFU counting was performed as described above, before and immediately after the challenge, in order to calculated percentage survival (Jan et al., 2001 (link)).