Profiling rhythmicity of bile salt hydrolase activity in the gut lumen with a rapid fluorescence assay

Diurnal rhythmicity of cellular function is key to survival for most organisms on Earth. Many circadian functions are driven by the brain, but regulation of a separate set of peripheral rhythms remains poorly understood. The gut microbiome is a potential candidate for regulation of host peripheral rhythms, and this study sought to specifically examine the process of microbial bile salt biotransformation. To enable this work, an assay for bile salt hydrolase (BSH) that could work with small quantities of stool samples was necessary. Using a turn-on fluorescence probe, we developed a rapid and inexpensive assay to detect BSH enzyme activity with concentrations as low as 6–25 μM, which is considerably more robust than prior approaches. We successfully applied this rhodamine-based assay to detect BSH activity in a wide range of biological samples such as recombinant protein, whole cells, fecal samples, and gut lumen content from mice. We were able to detect significant BSH activity in small amounts of mouse fecal/gut content (20–50 mg) within 2 h, which illustrates its potential for use in various biological/clinical applications. Using this assay, we investigated the diurnal fluctuations of BSH activity in the large intestine of mice. By using time restricted feeding conditions, we provided direct evidence of 24 h rhythmicity in microbiome BSH activity levels and showed that this rhythmicity is influenced by feeding patterns. Our novel function-centric approach has potential to aid in the discovery of therapeutic, diet, or lifestyle interventions for correction of circadian perturbations linked to bile metabolism.


II.
Experimental procedures S21-S23 a. General synthetic methods b. Synthetic procedures

IV.
Animal experiments and sample collection S27-28

V.
Procedures for detection of bacterial contamination in gnotobiotic mice S29 VI.

Statistical analyses S30
VII.         . ChoRhoS probe fluorescence is higher than CA-AMCA probe fluorescence when incubated with mouse fecal suspension. Two fecal samples (50 mg) were suspended in 1 mL of 1x PBS with and without beta-mercaptoethanol (BME) and incubated with both ChoRhoS and CA-AMCA probes at 37 °C. The reaction fluorescence, background fluorescence signal from the fecal suspensions, and probe blank fluorescence were quantified by measuring fluorescence at 495 nm and 525 nm for ChoRhoS and 350 nm and 450 nm for CA-AMCA, using a plate reader. A) ChoRhoS probe is cleaved in the presence and absence of BME. However, the probe turnover is much higher in the presence of BME. B) CA-AMCA probe is cleaved in the presence and absence of BME. However, background signal is significantly higher, and fluorescence turn-over is lower than probe blank. C) The S/N ratio is 3-fold higher for ChoRhoS than for CA-AMCA after 2 h. Sample+buffer and Sample+buffer+BME curves in figure A and B showed very low fluorescence and RFU plots overlapped on each other.       large intestine, and B) small intestine vs. cecum contents (gut contents were isolated from n=4 mice per group, separately, and analyzed separately). When incubated with the probe, fluorescence evolution from large intestine content (p=0.0008) and cecum (p=0.0012) was significantly higher than from small intestine content.    Whole-cell L. plantarum assay: A starter culture was generated by inoculation of 5 ml MRS broth from a L. plantarum colony grown on MRS agar. The starter culture was incubated at 37 °C overnight. The 5 mL starter culture was then added to 45 mL fresh MRS broth and grown until the S24 optical density reached 0.6 at 600 nm. The bacterial culture was centrifuged at 5000 rpm for 5 min. Then the media was removed and the cell pellet was resuspended in 15 mL 0.01M PBS and centrifuged. The cells were further washed with 15 mL PBS and centrifuged once more. The resulting pellet was suspended in 1000 μL PBS. The cell suspension was then serially diluted twofold to have different cell densities. 25 μL of each cell suspension and 25 μL 0.01M PBS buffer were added to Microfluor1 black flat bottom microtiter 96-well plates (Thermo Scientific). 50 μL solutions of probe (50 μM) in 0.01 M PBS with 0.42% DMSO were added to the cell suspensions in the plate to initiate the reaction. Reactions were immediately placed in a plate reader that was pre-warmed to 37 °C, and reaction progress was monitored at 495 nm and 525 nm excitation and emission wavelengths, respectively, for 20 min.
Whole-cell E. coli plate reader assay: A starter culture was generated by inoculation of 5 ml LB broth from an E. coli colony grown on LB agar. The starter culture was incubated at 37 °C overnight. The 5 mL starter culture was then added to 45 mL fresh LB broth and grown until the optical density reached 0.6 at 600 nm. The bacterial culture was centrifuged at 5000 rpm for 5 min. Assay with mouse fecal samples: Ice cold 0.01 M PBS with 20 mM beta-mercaptoethanol (BME) was added to fecal samples from SPF and GF mice (20 mg/mL for circadian rhythm analysis and 50 mg/mL for the rest of the experiments) and placed on ice for 30 min. The samples were extensively vortexed to obtain homogenized mixtures. After precipitation of large debris under gravity on ice, supernatant was filtered through a 35 µm cell strainer. Filtered fecal suspension was serially diluted with 0.01M PBS to test with different cell densities and 1:1 diluted fecal suspension was used for other experiments. 50 μL solutions of ChoRhoS probe (50 μM) in 0.01 M PBS with 0.42% DMSO were added to the diluted fecal suspensions (50 µL) in the plate to initiate the reaction. Reactions were immediately placed in a plate reader pre-warmed to 37°C, and reaction progress was monitored at 495 nm and 525 nm excitation and emission wavelengths respectively for 3 hours. All the reactions were done in triplicate.
For heat-shock experiments and BSH inhibitor experiments, fecal suspensions were pre-treated to lower the BSH activity with heat (95 °C) and a BSH inhibitor, caffeic acid phenethyl ester (CAPE; 200 µM), respectively, for 1 h prior to incubation with the probe.

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Human fecal sample analysis: Human fecal samples were obtained from a healthy donor. Homogenized human fecal sample (275 mg) was suspended in ice cold 0.01 M PBS with 20 mM BME to have the initial concentration of 25 mg/mL, and serially diluted to obtain different cell densities. For sample processing and plate reader measurements, similar procedure was followed as described above for mouse fecal sample analysis.

IV. ANIMAL EXPERIMENTS AND SAMPLE COLLECTION
Time-restricted and ad libitum feeding experiments: All the time-restricted and ad libitum feeding experiments were performed at Washington State University's Integrative Physiology and Neuroscience Behavioral Core. Experiments were conducted in accordance with Washington State University Institutional Animal Care and Use Committee (IACUC) protocol #6929. C57BL/6J mice were purchased from the Jackson laboratory and allowed to acclimatize to the local animal facility for 2 weeks before being used for experiments. The mice were group housed, between 2 and 3 mice per cage, and fed standard rodent chow #5001. In all experiments, 6 week-old male mice were used. Mice were 8 weeks of age at the beginning of measurements. Mice were housed under standard 12 h/12 h light/dark conditions (light phase = 05:30 to 17:30). Time-restricted feeding groups had access to food only during the light or dark phase, respectively, for 8 consecutive days. The ad libitum group had access to food all day. In each group, mice (n=4) were sacrificed by CO 2 asphyxiation for five time points at 6 h intervals over the period of one day (starting at the beginning of the dark phase), followed by organ collection and intestine content isolation. All the samples were collected in tubes, immediately frozen in liquid nitrogen upon collection, and stored at −80 °C until analysis. Germ-free mouse experiments: Mice were housed in the PNNL Animal Resource Center. All animal experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals (Battelle Richland IACUC approved protocol #2021-06). 7-8 weeks-old germ-free (GF) C57BL/6GF mice were purchased from Taconic Laboratory (German Town, NY). Male conventional C57BL/6N mice, which served as fecal microbiota transplantation (FMT) donors, were obtained from Taconic Laboratory (German Town, NY). GF mice were fed autoclaved LabDiet PMI 5010. Conventional mice were fed PMI lab Diet 5002. GF mice were group housed in a designated gnotobiotic facility room in a Tecniplast IsoP IVC caging system. Conventional mice were group housed in a separate room in Innovive IVC cages to maintain an environment similar to the GF mice. The rooms were kept at 22±2 °C and at 50±20% humidity on a 12 h/12 h light/dark cycle with ad libitum access to food and autoclaved water. Throughout the course of the experiment, mice stool samples were collected and tested for bacterial growth using aerobic and anaerobic culture conditions described in the next section. GF mice were randomly divided into two groups (n=3 each), which were a control group and an FMT recipient group. The FMT group was inoculated with fecal material from conventional mice via oral gavage. After FMT, both groups were housed for 2 weeks, and mice were sacrificed by CO 2 asphyxiation. Intestine samples were collected in tubes, immediately frozen in liquid nitrogen upon collection, and stored at −80 °C until further analysis. Fresh stool was collected from the male conventional C57BL/6N mice just prior to gavage. Collected fresh stool was pooled together and homogenized through vortexing in sterile 1x PBS as a 100 mg/mL slurry. The homogenized stool suspension was filtered through a 35 µm cell strainer. Mice were then colonized by oral gavage with 200 µL of filtered suspension of stool. FMT success was confirmed by culture methods identical to sterility determination for GF mice. Antibiotics experiments: 5-9-weeks-old male conventional C57BL/6N mice were kept under 12 h/12 h light/dark cycle with ad libitum food access (PMI lab Diet 5002) at 22±2 °C temperature and at 50±20% humidity. Mice were randomly divided into two groups (n=4 each). They were given oral gavage of antibiotics (antibiotic group) or water (vehicle group) every 12 h. The

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antibiotic cocktail was comprised of four antibiotics and one antifungal and was made as follows: ampicillin (100 mg/kg), metronidazole (100 mg/kg), neomycin (100 mg/ kg), vancomycin (50 mg/kg), and amphotericin B (1 mg/kg). Amphotericin B was added to prevent fungal overgrowth or opportunistic infections. This cocktail was made fresh every 36 h, aliquoted for each dose, and stored at -20 C.

V. PROCEDURES FOR DETECTION OF BACTERIAL CONTAMINATION IN GNOTOBIOTIC MICE
Mouse fecal sample collection and handling was performed inside a clean biosafety cabinet (BSC) in a sterile manner. Feces were most commonly collected during cage changeout or other planned procedure/manipulation of germ-free mice inside the BSC. Fecal samples were collected as fresh as possible and collected directly from the anus using sterile tweezers/forceps and placed in a sterile pre-labeled screw-cap vial.
Culturing activities were performed in a clean BSC in a sterile manner. Trypticase™ soy agar with 5% sheep's blood culture plates were used for aerobic cultures, Brucella agar plates containing 5% sheep blood, hemin and vitamin K were used for anaerobic cultures, and thioglycolate growth media was used for both aerobic and anaerobic growth.
A sterile swab was premoistened with sterile 1x PBS to prevent the feces from sticking to the swab. The sample vial containing the feces was opened and the premoistened sterile swab was used to transfer a small amount of smeared sample. The thioglycolate growth media tubes were inoculated and placed in a 37 °C incubator. The same swab with feces that was used to inoculate the thioglycolate growth media was used to smear on half the surface of the blood agar plates. Using a new premoistened sterile swab, a small amount of fecal sample was transferred onto the surface of the appropriate culture plate and streaked out, spreading out over the second half of the surface of the plate. Brucella agar plates containing 5% sheep blood, hemin and vitamin K were placed in a GasPack™ EZ anaerobe gas generating pouch system, following manufacturer's instructions. The pouch containing the Brucella agar plates and the Trypticase™ soy agar with 5% sheep's blood culture plates was placed in a 37 °C incubator. The cultures were checked at 24, 48, and 72 h, and incubation was followed by 5-7 days at room temperature.

VI. STATISTICAL ANALYSES
For the analysis of rhythmic oscillations, the relative fluorescence units (RFU) values for fecal samples after 84 min co-incubation with the probe were plotted against the time of collection. To analyze diurnal rhythms, the data points were subjected to cosinor analysis (i.e., fitted to a cosine function with a fixed period of 24 h) using non-linear regression. 2 Amplitude was tested against zero (t test) to confirm statistical significance of rhythmicity, and peak timing was compared between experimental conditions (F test) to assess phase differences. For other analyses, pairwise comparisons were performed with Student's t test using GraphPad Prism software.          Figure S28. LRMS spectrum for N-acetylrhodamine. S36