Taurocholate

Eight compounds with known pharmacokinetic parameters in human brain were selected: befloxatone (Synthélabo), flumazenil (Sigma), raclopride (tartrate salt, Sigma), erlotinib (Fluorochem), verapamil (Sigma Aldrich), buprenorphine (Sigma Aldrich), 2F-A85380-tartrate (Eras Labo) and loperamide (Sigma). In addition, eleven other compounds with diverse physicochemical properties were selected. We tested six non-radiolabeled compounds: dextromethorphan (hydrobromide monohydrate, Sigma Aldrich), levofloxacin (Sigma), sulfasalazine (Sigma), caffeine (Sigma Aldrich), taurocholate (sodium salt, Sigma), Lucifer Yellow (CH dilithium salt, Sigma); and five radiolabeled compounds: [U−¹⁴C]-sucrose (molar activity: 601 mCi.mmol⁻¹), [³H]propranolol (molar activity: 24 Ci.mmol⁻¹), [³H]verapamil hydrochloride (molar activity: 85 Ci.mmol⁻¹), [³H]vinblastine sulfate (molar activity: 10.3 Ci.mmol⁻¹), [³H]digoxin (molar activity: 40.0 Ci.mmol⁻¹) were purchased from Perkin Elmer (Massachusetts, SA).

ITC measurements were carried out at 20 °C with a Nano ITC Standard Volume isothermal calorimeter (TA Instruments, New Castle, DE) controlled by the ITCRun software. The N-terminal His₆ tags of all purified proteins were removed before ITC analysis. For titrations with DPD/AI-2 (Omm Scientific), the tag-free proteins were dialyzed against a Tris buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) and diluted to 70 μM, while DPD/AI-2 was diluted with the same buffer to 700 μM. For titrations with 100 μM taurocholate, taurodeoxycholate (both Sigma-Aldrich), SicA, and its variants, the tag-free proteins subjected to the sample cell were dialyzed against the Tris buffer and diluted to 10 μM. Protein samples were dialyzed against the buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 5 mM MgCl₂, and diluted to 10 μM for titrations with 100 μM c-di-GMP, c-di-AMP, or cGMP dissolved in the same buffer. After being degassed, 1 ml of the protein and 250 μl of the ligand solution were added to the sample cell and the syringe, respectively. There were 25 injections per experiment and the stirring speed was 200 rpm. In control experiments, the ligand solution was titrated into the buffer in the sample cell to obtain the heat of dilution. Microcalorimetric data were corrected by subtracting the heats of dilution and fit to the independent binding site model using the NanoAnalyze software.

Standards for primary bile acids cholate (CA) and chenodeoxycholate (CDCA), conjugated primary bile acids glycocholate (GCA), taurocholate (TCA), glycochenodeoxycholate (GCDCA), and taurochenodeoxycholate (TCDCA), secondary bile acids lithocholate (LCA), deoxycholate (DCA), ursodeoxycholate (UDCA), and hyodeoxycholate (HDCA), and conjugated secondary bile acids glycolithocholate (GLCA), taurolithocholate (TLCA), glycodeoxycholate (GDCA), and taurodeoxycholate (TDCA) were purchased from Sigma.

Stool samples from patients with diarrhea, and suspected of having CDI, were tested in the diagnostic laboratory for C. difficile using the GeneXpert C. difficile epi test following manufacturer’s instructions [3 (link), 17 ]. C. difficile cultures were performed in the research laboratory. Briefly, stool samples were mixed with an equal volume of 100 % ethyl alcohol for 60 min at room temperature. One hundred microliters of the mixture were spread on Clostridium difficile base agar plates (CDBA) (Oxoid, Inc), supplemented with 0.1 % taurocholate (Sigma), norfloxacin 12 mg/L, moxalactam 32 mg/L, Cystein-HCl 0.5 g/L and 7 % defibrinated horse blood and incubated at 36 ± 0.5 °C for 72 to 96 h under anaerobic conditions (90 N₂, 5 CO_2, and 5 H₂). Presumptive identification as C. difficile was based on colony size, morphology and fluorescence properties [18 (link)]. Isolated single C. difficile colonies were picked and inoculated into 0.5 mL of pre-reduced Chopped Meat Glucose Broth (CMGB) (Anaerobic Biosystems, USA) and incubated for 72 to 96 h under anaerobic conditions. Bacteria and spores were collected from the broth medium by centrifugation at 13,000xg for 5 min, and the DNA was extracted by boiling the sediment in 1 % Chelex-100 resin (Sigma, Inc) suspension for 20 min. Samples were centrifuged at 13000xg for 5 min, and the supernatant was used for downstream testing.

To enumerate spores, overnight cultures of C. difficile grown in BHIS medium supplemented with thiamphenicol, where necessary, were used to inoculate 10 ml of fresh BHIS medium. At intervals, samples of culture were taken and divided. To determine the total number of colony-forming units, one sample was serially diluted and plated on BHIS medium plus 0.1% taurocholate (Sigma). To determine the number of spores, the second sample was heat-killed by incubation for 25 min at 65 °C prior to plating on BHIS medium plus 0.1% taurocholate. The number of spores was subtracted from the total cell counts to give the vegetative cell numbers.
To analyze cell length, C. difficile strains were grown in BHIS broth at 37 °C in an anaerobic chamber for 12 h. 0.5 ml of each culture was centrifuged at 4000 × g for 2 min. Following gentle resuspension in 150 μl of 1× PBS, 3 μl of culture was placed on top of 1.2% agarose pads and left to dry for 15 min. Slides were visualized using an Eclipse E600 microscope (Nikon), using a Plan Fluor ×100 objective lens, using oil immersion. Images were captured using a Retiga 2000R camera, using the QCapture Pro Software (QImaging), and images were processed using the ImageJ software.

C. difficile strains R20291 and R20291ΔtcdR were used in this study (35 ). C. difficile spores were maintained on brain heart infusion (BHI) medium supplemented with 100 mg/L l-cysteine and 0.1% taurocholate (Sigma-Aldrich, T4009). Cultures were started by inoculating a single colony from the plate into BHI liquid medium supplemented with 100 mg/L l-cysteine and grown anaerobically. Caco-2 (ATCC, HTB-37), HCT116 (ATCC, CCL-247), IM90 (ATCC, CCL-186) and Vero cells (ATCC, CCL-81) were cultured in DMEM supplemented with 2 mm l-glutamine and 10% FBS and incubated in 5% CO2 at 37°C. CellEvent Caspase-3/7 Green Detection Reagent (C10723) and PrestoBlue Cell Viability Reagent (A13261) were purchased from ThermoFisher Scientific. All bile acids were dissolved in DMSO to a final concentration of 40 mM. Vendors and catalog numbers of bile acids used in this study are listed in Table S1 in the supplemental material. Staurosporine (S6942) was purchased from MilliporeSigma. Commercially available C. difficile toxins (A and B) and antitoxin for the Vero cell assay were purchased from List Biological Labs (Toxin A:152C and Toxin B: 155C) and TechLabs (T5000), respectively. The plasmid pDSW1728-PtcdA::mCherry was a kind gift from Dr. Craig Ellermeier (University of Iowa). All strains, plasmids, and primers used in this study are listed in Table S2.