Characterization of interactions of dietary cholesterol with the murine and human gut microbiome

Chemicals

All HPLC solvents were purchased from Fisher Chemicals as Optima LC–MS grade. 27-Alkyne cholesterol (Chol^Alk) was purchased from Click Chemistry Tools. Sodium cholesteryl sulfate (Chol^Sulf), glycocholic acid, glycoursodeoxycholic acid, cholic acid, chenodeoxycholic acid and lithocholic acid were purchased from Sigma-Aldrich. Cholesterol was purchased from MP Biomedicals. Olive oil was purchased from Spectrum Chemical.

Single-species bacterial cultures supplemented with cholesterol or Chol^Alk

Cholesterol, Chol^Alk and bile acid stocks were prepared as 25 mM solutions in ethanol. Bacteria were cultured in their respective media to stationary phase. Then 1 ml of the bacterial stock was diluted in 25 ml of fresh media containing 10 μM of the respective small molecule for 48 h at 37 °C. E. coprostanoligenes ATCC51222 was cultured in BCM without cholesterol as described in Kenny et al.¹⁷. Bacteroides species were cultured in minimal media as described in Lee et al.²¹. A. caccae and Ruminococcaceae species (TSD-27) were cultured with ATCC medium 2971. Bifidobacterium species were cultured in BSM broth (Sigma-Aldrich). Lactobacillus species were cultured in MRS broth (Sigma-Aldrich). BL21 E. coli was cultured in M9 minimal media with glucose. Except for E. coli, all bacterial cultures were grown in glass bottles under anaerobic conditions. E. coli cultures were grown in sterilized Erlenmeyer flasks with shaking at 250 r.p.m. All bacterial pellets were collected at 4,000g for 20 min at 4 °C in 50 ml centrifuge tubes, the supernatant was aspirated, and pellets resuspended in 1 ml of PBS. The bacteria were then moved to 1.5 ml centrifuge tubes and pelleted again. The supernatant was removed and the pellet was washed once more with PBS. The spent PBS was removed, the pellet was flash-frozen using liquid nitrogen, and dried via lyophilization until further processing.

Animal experiments

All mouse experiments were performed according to a protocol approved by the Cornell University Institutional Animal Care and Use Committee (protocol no. 2010-0065).

In vivo dietary cholesterol uptake

Female conventionally raised excluded flora Swiss Webster mice were purchased from Taconic Biosciences at 5 weeks of age and were acclimated for at least 5 d before undergoing any procedure. For sequencing experiments, 12 mice were preweighed and randomly assigned to one of four treatment groups: (1) +cholesterol, (2) +Chol^Alk, (3) +vehicle and (4) no gavage. Mice were housed three per cage with three mice per treatment group. For comparative metabolomics experiments, 12 mice were preweighed and randomly assigned to one of three treatment groups: (1) +cholesterol, (2) +Chol^Alk and (3) +vehicle. Mice were housed four per cage with between two and four mice per treatment group. Mice were housed in a climate-controlled environment with 12 h light and dark cycles and reared on a standard sterilized breeder diet (LabDiet 5021) with ad libitum access to autoclaved water. For the dietary cholesterol treatment as a part of the BOSSS workflow, mice received 100 μl of cholesterol or Chol^Alk at 20 mg per kg body weight in olive oil as a vehicle via oral gavage using a 20 Gauge gavage needle (Fine Science Tools). Mice were gavaged once daily with either of the two cholesterol treatments, vehicle control or no gavage for 7 d. On the day they were killed, mice received the final gavage and were then fasted for 5 h before euthanasia via decapitation. Caecal contents were collected, snap-frozen using liquid nitrogen, and stored at −80 °C until further processing. This experiment and subsequent analysis were repeated and representative metagenomic data are displayed in Fig. 2.

Isolation and fixation of microbial cells from caecal content samples

The general procedure to isolate microbial cells from caecal content samples was modified from our workflow demonstrated in Lee et al.²¹. In brief, the thawed caecal content samples were preweighed and diluted ten times with ice-cold sterile 1× PBS in a 2-ml screw-top tube. Samples were then subjected to intermittent vortex for 5 min followed by mild sonication for 20 s total on time, with alternating 2 s on pulses, and 2 s off pulses, at 3–6 W (Qsonica Ultrasonic Processor, model Q700, with a water bath adaptor, model 431C2). Samples were centrifuged at room temperature for 2 min at 200g, and supernatants were transferred to a fresh 1.5 ml tube. The above steps were repeated twice on the remaining cell pellets for maximizing cell recovery and the supernatants were pooled and centrifuged at 18,000g for 10 min at room temperature. The bacterial pellets were washed three times with ice-cold 1% BSA/PBS. Cell pellets were resuspended in 10% buffered formalin for 10 min. Cells were then washed and incubated with 0.1% Triton X-100 in PBS for 30 min at room temperature on an end-over-end rotor for cell permeabilization and removal of non-assimilated free Chol^Alk.

Copper(I)-catalysed azide-alkyne cycloaddition staining (click reaction)

Bacterial cells containing Chol^Alk derivatives were detected and labelled with AF647-azide using freshly prepared click reaction cocktail following the manufacturer’s instructions for the Click-&-Go Click Chemistry Reaction Buffer Kit (Click Chemical Tools) at a final fluorophore concentration of 5 μM for 1 h at room temperature and in the dark. Cell pellets were washed five times and resuspended with 1% BSA/PBS to remove any non-specific fluorescent signals before fluorescence imaging and flow cytometry.

Isolation of live gut microbes from mice faeces

Freshly collected faecal pellets from untreated mice receiving a chow diet were weighed and put into a screw-top tube filled with 250 μl of prewarmed (37 °C) and prereduced sterile PBS with 0.1% l-cysteine. For flow cytometry, the faecal microbes were isolated following the same procedure as the preparation steps for caecal microbial cells described above. For ex vivo cultures mentioned below, the samples were vortexed for 5 min and brought into an anaerobic chamber (gas mix: 70% N₂, 25% CO₂, 5% H₂). The suspension was left standing for 5 min to precipitate the insoluble particles, and the supernatant portion was transferred to a fresh prereduced tube for the subsequent ex vivo culture.

Ex vivo cultures of live faecal microbes for medium selection

A 50 μl aliquot from the isolated faecal microbial samples was used to inoculate 25 ml of four different media formulations: GMM (developed by Li et al.²⁸), modified Gifu Anaerobic medium (HyServe), BCM and Brain Heart Infusion. Cultures were incubated at 37 °C in an anaerobic chamber for 2 d, and the cell pellets were harvested by centrifugation at 4,000g for 30 min at 4 °C.

Ex vivo culture of live faecal microbes with cholesterol or Chol^Alk

After identifying the top two candidate medium to culture cholesterol-interacting bacteria, 50 μl of the isolated faecal microbial samples was inoculated into 25 ml of the selected media with 10 μM cholesterol or Chol^Alk for 48 h under anaerobic conditions at 37 °C.

FACS gate optimization

To optimize the capture of Chol^Alk-interacting microbes, E. cop, a bacterium active in cholesterol metabolism¹⁷, was selected to establish the positive gate (Alk+). E. cop was cultured with Chol^Alk for 3 d (E. cop^Alk), followed by AF647-azide detection as described above. The purity of the positive sort was further confirmed by both fluorescence imaging and targeted metabolomics. Unlabelled faecal microbiota were used for development of the negative gate (Alk–). To improve stringency of the gates, both E. cop^Alk and faecal microbes were first stained with 1 μg ml⁻¹ Hoechst 33342 (Invitrogen) to normalize cell counts via a Leica DM500 fluorescence microscope according to Hedal et al.⁶⁸. Before flow cytometry, the normalized cell suspensions of E. cop^Alk and faecal microbes were pooled at varying ratios and analysed using a BD Biosciences Melody Sorter. Data were acquired using BD FACSChorus RUO software v.2.0 and analysed using FlowJo v.10.6.2 software. The BD Biosciences Melody Sorter was sterilized by runs of 70% ethanol, and the instrument was kept under sterile conditions during sorting using sheath fluid prepared with sterile PBS. Samples were passed through a 35 µm nylon mesh cell strainer (Corning), the AF647-azide dye was excited using a 640-nm red laser and fluorescence was captured with a 660 nm/20 nm filter. Gates were established and adjusted following the procedures as described in Lee et al.²¹.

FACS to identify Chol^Alk-containing bacteria

FACS was performed as described in Lee et al.²¹ via the established gates (vida supra). Some 60,000 events in each gate with an AF647 signal greater than ~10³ fluorescent intensity (Alk+) or 10² to 10⁻² fluorescent intensity (Alk–) were captured and segregated into two separate 5-ml round-bottom tubes (BD Biosciences) containing 1% sterile BSA/PBS. A second round of sorting was performed on both the Alk+ and Alk– fraction with the parameters above to ensure the purity of the cells. After sorting, cells were transferred to 1.5 ml tubes and centrifuged at 18,000g for 10 min at 4 °C. The supernatant was discarded, and the sorted cells were counterstained with 1 μg ml⁻¹ Hoechst 33342 (Invitrogen), then washed and resuspended with 1% BSA/PBS for imaging or sequencing.

Fluorescence microscopy

Resuspended cells were mounted onto glass slides with Vectashield Vibrance mounting media (Vector Labs) and analysed using a Leica DM500 fluorescence microscope. All the images were acquired using LAS X v.3.6 software and analysed using Fiji Image J software as described in Schindelin et al.⁶⁹. In addition, the supernatant from each microbial isolation from caecal contents was imaged for confirmation of the absence of bacteria with Hoechst 33342 (1 μg ml⁻¹) and imaged using fluorescence microscopy. Samples were imaged by using two filters: UV (359 nm/461 nm) for Hoechst 33342, and Cy5 (650 nm/670 nm) for AF647 to detect the presence of alkyne-containing metabolites.

16S rRNA gene amplicon sequencing and analysis

Genomic DNA was isolated from the microbial cell pellets as described previously in Lee et al.²¹. The amplicon libraries were created by PCR amplification of the V4 variable region using primers with common adaptor sequences: 515F and 806R⁷⁰. Barcoded reverse and non-barcoded forward primers were used with Taq DNA polymerase Master Mix (TONBO Biosciences) according to the manufacturer’s directions. Samples were amplified in duplicate with the following thermocycler protocol: hold at 94 °C for 3 min; 30 cycles of 94 °C for 45 s, 50 °C for 1 min, 72 °C for 1.5 min; and hold at 72 °C for 10 min. The duplicate final amplified products were pooled. 16S rRNA gene amplicons were cleaned using Mag-Bind RxnPure Plus beads (Omega Bio-tek). Samples were mixed in equimolar amounts before sequencing on Illumina’s MiSeq platform using 2 × 250 bp paired-end runs. Sequence data are available in the National Center for Biotechnology Information (NCBI) sequence read archive under BioProject PRJNA718322.

Sequence data processing was performed using the QIIME 2 (v.2020.2) pipeline⁷¹. The DADA2 method²⁷ was applied to quality-filter sequences and categorize ASVs. The resulting ASVs were assigned taxonomy by mapping with the SILVA 132 database⁷². The QIIME output data were imported to RStudio (v.1.0.136) with the Bioconductor package phyloseq⁷³ for normalizing and plotting the input data. The volcano plot for identifying differentially abundant taxa between the two sorted fractions was analysed and plotted via DESeq2 (ref. ⁷⁴) and EnhancedVolcano⁷⁵ respectively. Fold-enrichment analysis for each ASV in the sorted population was performed according to Ronda et al.⁷⁶. This involved calculating the relative abundance of ASVs in the unsorted population as the normalized number of reads in a sample, and the fold-enrichment of each ASV in the Alk+ and Alk– populations was further calculated as the relative abundance in the sorted population divided by the relative abundance in the unsorted total population.

Shotgun metagenomic library construction and sequencing

To minimize potential contamination from eukaryote DNA (mice and human), both a host depletion and a microbial DNA enrichment step were conducted before Illumina library preparation with the HostZERO Microbial DNA Kit (Zymo Research). Purified DNA was quantified using the Qubit dsDNA High-Sensitivity Assay Kit (Invitrogen). The sequence libraries were prepared using the NexteraXT DNA Library Preparation Kit (Illumina) followed by size and quality assessment using a Bioanalyzer (Agilent). After quantification with the Qubit 3.0 fluorometer, libraries were pooled and sequenced on an Illumina NextSeq 500 using a paired-end 2 × 150 bp protocol. Sequence data are available in the NCBI sequence read archive under BioProject PRJNA718322.

The raw read data were first processed using Kneaddata⁷⁷ (v.0.6.1). Briefly, this module includes quality trimming (with 4-mer windows with mean Phred quality <25) by Trimmomatic⁷⁸ and host-derived sequence removal by mapping with bowtie2 against the reference genome⁷⁹. For taxonomic classification, Kraken2 was used to assign taxonomy to the Kneaddata-filtered reads⁸⁰, and the resulting assigned taxonomy was further subjected to Bracken2 for estimating the species abundance⁸¹. The resulting kreport files were converted to MetaPhlAn-compatible file using KrakenTools⁸⁰ for the subsequent establishment of taxonomic and phylogenetic trees using GraPhlAn pipeline⁸². For functional annotation, we applied the Human Microbiome Project Unified Metabolic Analysis Network 3 (HUMAnN3) pipeline that maps reads to functionally annotated organism genomes and uses a translated search to align unmapped reads to UniRef90 protein clusters⁸³. Also, to focus on the functional expression of the Alk+ fraction of interest, the gene family clusters were determined using Welch’s two-sided t-test followed by Benjamin–Hochberg FDR correction (Supplementary Table 4). Only clusters identified as significantly abundant in the Alk+ fraction were retained for plotting. The unstratified gene family abundances were further converted to Gene Ontology terms for assessing the general metabolic activities⁸⁴. The detailed HUMAnN workflow is published in Franzosa et al.⁸⁵.

Heterologous expression and in vivo activity of cholesterol sulfotransferase candidates

Cholesterol sulfotransferase candidates were selected from BLAST analysis of the B. thetaiotaomicron genome with the amino acid sequences of human SULT2A1 and SULT2B1. The six candidates were then amplified from the genome of B. thetaiotaomicron with the primers listed in Supplementary Table 1 and cloned into pET28a E. coli expression vector with the NdeI and BlpI cut sites. Ligated plasmid was transformed into TOP10 E. coli and screened via colony PCR for positive insertions. Positive pET28 sulfotransferase plasmids were purified with the QIAPrep Spin Miniprep Kit (Qiagen) and were verified via Sanger sequencing. Confirmed plasmids were transformed into BL21 E. coli (New England Biolabs) and selected with Luria broth (LB) plates containing 50 μg ml⁻¹ kanamycin. An individual colony of each pET28 sulfotransferase transformed candidate was picked into separate 5 ml of LB media and cultured overnight at 37 °C in 15 ml plastic culture tubes with shaking at 250 r.p.m. Some 50 μl of the overnight cultures were used to inoculate 6 ml of M9 media containing glucose treated with either cholesterol or Chol^Alk. No isopropylthiogalactoside was needed because expression from the T7 promoter is leaky. After shaking at 37 °C 250 r.p.m. for 18 h, cells were centrifuged at 4,000g for 20 min at 4 °C and the spent media was removed. The bacterial pellets were resuspended in 1 ml of PBS and moved to 1.7 ml centrifuge tubes. Samples were centrifuged again at 4,000g and 4 °C for 10 min and the PBS removed. Samples were frozen with liquid nitrogen, lyophilized to dryness and underwent metabolite extraction and LC–MS analysis as described below.

Immobilized metal-affinity chromatography enrichment and in vitro activity of polyhistidine-tagged BT_0416 from E. coli

BL21 E. coli containing pET28 Bt_0416 plasmid was grown in 10 ml of LB overnight. The 10 ml of overnight culture was then diluted into 1 l of terrific broth containing kanamycin and cultured at 37 °C in a 2 l Erlenmeyer flask rotating at 250 r.p.m. Once an optical density at 600 nm (OD₆₀₀) of 0.6 was reached, culture temperature was reduced to 20 °C, isopropylthiogalactoside was added to a final concentration of 100 μM and cultured for an additional 16 h. Cells were harvested at 4,000g for 12 min at 4 °C and resuspended in 80 ml of lysis buffer (20 mM sodium phosphate pH 7.4, 300 mM sodium chloride, 1× ProBlock Gold Bacteria 2D Protease Inhibitor Cocktail; Gold Biotechnology). The slurry was sonicated with a probe sonicator and then centrifuged again at 20,000g for 20 min at 4 °C. The supernatant was applied to pre-equilibrated Ni-NTA (Gold Biotechnology). The lysate was gravity fed through the beads and the beads were washed with 15 ml of lysis buffer. The captured protein was eluted with 15 ml of lysis buffer containing 250 ml of imidazole and 10% glycerol. The eluent was concentrated to 500 μl with an Amicon Ultra-15 30 K distinct molecular weight cutoff spin filter (Merck Millipore) and flash-frozen over liquid nitrogen until further analysis. Some 5 μl of partially isolated BT_0416 (Supplementary Fig. 8) was added to a 100 μl scale reaction solution containing 50 mM potassium phosphate pH 7.5, 250 μM cholesterol, 250 μM PAPS (Sigma-Aldrich) and 0.2% Triton X-100 and incubated at 37 °C for 12 h. Solutions were dried with a SpeedVac Vacuum Concentrator. Samples then underwent metabolite extraction and LC–MS analysis as described below.

BT_0416 knockout strain (BtΔtdkΔ0416)

Mutagenesis was performed as described in Johnson et al.⁸⁶. Specifically, to generate an in-frame deletion of Bt_0416 in B. thetaiotaomicron, the strain B. thetaiotaomicron VPI-5482 tdk was used⁸⁷. Two 700 bp regions, each flanking the gene to be deleted, were PCR-amplified (NEB Q5 Hot Start High-Fidelity DNA Polymerase) and cloned into EcoRV-HF and NotI-HF linearized pExchange_tdk. Primers are listed in Supplementary Table 1 and strains are listed in Supplementary Table 2. The assembled construct was transformed into E. coli S17-1 λpir (Biomedal), plated on LB agar–streptomycin–carbenicillin plates, and transformants screened for incorporation of the plasmid. Final concentrations of antibiotics and selection agents were as follows: erythromycin 25 μg ml⁻¹, gentamicin 200 μg ml⁻¹, streptomycin 100 μg ml⁻¹, carbenicillin 100 μg ml⁻¹, 5′-fluoroxyuridine (FUdR) 200 μg ml⁻¹. To conjugate cells, recipient and donor cells were inoculated from overnight cultures (B. thetaiotaomicron tdk at 1:1,000; E. coli transformant at 1:250) and grown to early exponential phase (OD₆₀₀ = 0.2–0.3), at which time the donor and recipient strains were combined in a 1:1 ratio and centrifuged for 20 min at 3,220g at room temperature. The bacterial pellet was resuspended in 100 μl of brain heart infusion broth-supplemented with 5 g l⁻¹ yeast extract and 5 μg ml⁻¹ hemin (BHIS), plated as a puddle on BHIS–10% defibrinated sheep blood agar plates, and incubated anaerobically at 37 °C for 20 h. The conjugation puddle was then scraped, serially diluted in PBS and incubated aerobically at 37 °C on BHIS–10% defibrinated sheep blood–gentamicin–erythromycin agar plates. Colonies were screened for merodiploids via PCR, cultured overnight in liquid BHIS and serially diluted onto BHIS–10% defibrinated sheep blood–gentamicin–FUdR agar plates. Colonies were PCR screened for deletion of the gene and confirmed via Sanger sequencing and metabolomics.

Phylogenetic tree of Bt_0416-like genes in various organisms

A BLASTP analysis was performed utilizing the BLAST tool from the Kyoto Encyclopedia of Genes and Genomes (https://www.kegg.jp/) tool set. The top 50 results were selected and the TREE function was applied. Alignment and phylogenetic reconstructions were performed using the function ‘build’ of ETE3 v.3.1.1 (ref. ⁸⁸). Alignment was performed with MAFFT v.6.861b using the default options⁸⁹. The tree was constructed using FastTree v.2.1.8 with default parameters⁹⁰.

Monocolonization and conventionalization of GF mice

Forty 5-week-old female GF Swiss Webster mice were purchased from Taconic Biosciences. Mice were randomly assigned to one of the four groups: (1) GF; (2) WT, BtΔtdk mono-associated; (3) KO, BtΔtdkΔ0416 mono-associated; and (4) CONV-D, conventionalized (three cages per group, and three or four mice per cage). For monocolonization experiments, colonization was achieved by a single oral gavage with 10⁹ c.f.u. of either BtΔtdk (cholesterol sulfotransferase competent) or BtΔtdkΔ0416 (cholesterol sulfotransferase null) in 200 μl of sterile PBS. Mice were maintained monocolonized in sterile cages on a Rodent NIH-31 Modified Diet (Zeigler) and were given access to sterile autoclaved water ad libitum. For conventionalization, 1 g of faecal pellets were freshly collected from specific pathogen-free Swiss Webster mice (Taconic Biosciences) and resuspended in 15 ml of sterile PBS supplemented with 0.1% (w/v) l-cysteine hydrochloride, the faecal slurry then sat at 37 °C anaerobically for 5 min, the resulting supernatant was collected and 200 μl of the suspension per mouse was introduced to a GF mouse via oral gavage. Two weeks post-colonization, faecal pellets were collected, and mice were euthanized by decapitation. Blood was collected, with 300 μl of blood mixed with 250 µg ml⁻¹ freeze-dried heparin for the whole-blood sample, and the rest was equilibrated at room temperature for 1.5 h, followed by centrifugation at 3,000 r.p.m. for 15 min to retrieve serum samples. Caecal content was collected, snap-frozen in liquid nitrogen, and stored at −80 °C.

Evaluation of Bacteroides colonization

Faecal pellets from each group of mice were weighed, mashed and vortexed in 1 ml of sterile PBS, and diluted to plate c.f.u. on triplicate Brain Heart Infusion agar plates supplemented with 10% defibrinated horse blood. Plates were placed in a 37 °C incubator inside an anaerobic chamber (Coy Laboratory Products). Plates were removed after 48 h and colonies were counted manually.

Evaluation of bacterial load

Bacterial load was determined by real-time qPCR using a protocol modified from Gomes-Neto et al.⁹¹. In brief, an aliquot of caecal content was resuspended with 750 μl of lysis buffer (200 mM NaCl, 100 mM Tris pH 8.0, 20 mM EDTA, 20 mg ml⁻¹ lysozyme), and transferred to a sterile screw-top tube containing ~500 mg of 0.1 mm zirconium beads (BioSpec Products). After extraction at 350 r.p.m. and 37 °C for 30 min, 85 μl of 10% SDS solution and 40 μl of proteinase K (15 mg ml⁻¹, Qiagen) were added, and the samples incubated for 10 min at 55 °C. Samples were then homogenized in a Mini-BeadBeater (BioSpec) for 1 min at maximum speed. After cooling on ice for 3 min, 500 μl of phenol–chloroform–isoamyl alcohol (25:24:1, Sigma-Aldrich) were added, and tubes were inverted ten times until there was more or less no layer separation, followed by centrifugation at 18,000g for 5 min. The aqueous layer was transferred to a new sterile microcentrifuge tube containg the same volume of chloroform–isoamyl alcohol (24:1, Sigma-Aldrich). After centrifugation at 18,000g for 5 min, the final aqueous layer was transferred to a new sterile microcentrifuge tube and 0.6 vol. of ice-cold isopropanol was added. After overnight precipitation at −20 °C, DNA was recovered by centrifugation at 18,000g for 30 min at 4 °C, followed by three rounds of 75% nuclease-free ethanol washing. The final DNA pellet was resuspended in 100 μl of nuclease-free water. Bacterial DNA was amplified with universal 16S primers as listed in Supplementary Table 1 using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) and QuantStudio 7 real-time PCR system (Applied Biosystems). The results were normalized to caecal content weight.

Metabolome extraction

One millilitre of methanol was added to the dried material and sonicated for 3 min, with on/off cycles of 3 s on, 2 s off, at 100% power on a Qsonica Ultrasonic Processor with the Cup Horn water bath attachment maintained at 20 °C. The samples were then placed on an end-over-end rotator and metabolites were extracted overnight. Samples were then centrifuged at 18,000g at 4 °C for 30 min. The clarified supernatant was collected and transferred to a fresh 1.7 ml centrifuge tube. The collected extracts were evaporated to dryness using a SpeedVac vacuum concentrator (Thermo Fisher Scientific) and reconstituted in 200 µl of methanol. Samples were sonicated again and centrifuged at 18,000g at 4 °C for 30 min. In total, 150 µl of clarified concentrated extracted metabolome was transferred to an HPLC vial utilizing an insert (Thermo Fisher Scientific) and stored at 4 °C until LC–MS analysis.

LC–MS analysis for untargeted metabolomics

High-resolution LC–MS analysis was performed on a Thermo Fisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a heated electrospray ionization (HESI) ion source. Five microlitres of concentrated extract was injected and separated using a water–acetonitrile gradient on an Agilent Technologies InfinityLab Poroshell 120 EC-C18 column (50 mm × 2.1 mm, particle size 2.7 μm, part no. 699775-902) maintained at 50 °C. Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. The A/B gradient started at 20% solvent B for 1 min after injection, increased linearly to 100% solvent B at 16 min and was held at 100% solvent B for 5 min, at a flow rate 0.6 ml min⁻¹. Mass spectrometer parameters were as follows: spray voltage, 3.5 kV (positive mode) and 3.0 kV (negative mode); capillary temperature, 380 °C; prober heater temperature, 400 °C; sheath, auxiliary and spare gas of 60, 20 and 2, respectively; S-lens RF level 50, resolution 240,000 at m/z 200, AGC target 3 × 10⁶. Each sample was analysed in positive and negative modes with an m/z range of 150–800.

Untargeted metabolomic analysis

Untargeted metabolomic analysis RAW files generated from HPLC–HRMS acquisitions were converted to mzXML files utilizing MSconvertGUI software (proteowizard.sourceforge.net)⁹². Differential molecular features were determined by subjecting mzXML files to Metaboseek Software v.0.9.6 (metaboseek.com) utilizing the XCMS package^42,44. Differential features were filtered using the minFoldOverCtrl, minInt and Fast_Peak_Quality filters, and then curated manually to remove adducts and isotopes (Supplementary Tables 5 and6). The curated features were assigned molecular formulas and then subjected to MS/MS. The MS/MS fragments were also assigned molecular formulas and structures were inferred. A focus was placed on structures that could be inferred.

Metabolomes were compared between caecal contents of mice orally exposed to Chol^Alk, cholesterol or vehicle control (olive oil gavage) in the in vivo condition (Supplementary Table 5). In the ex vivo condition, metabolomes were compared between faecal cultures grown in multiple media conditions (media A, media B), these conditions plus ethanol as a vehicle control (media A + ethanol, media B + ethanol), these conditions supplemented with cholesterol (media A + cholesterol, media B + cholesterol) and these media conditions supplemented with Chol^Alk (media A + Chol^Alk, media B + Chol^Alk) (Supplementary Table 6).

LC–MS analysis for targeted metabolomics

Targeted LC–MS analysis was performed on a Thermo Scientific Vanquish Horizon UHPLC System coupled with a Thermo Scientific TSQ Quantis Triple Quadrupole mass spectrometer. Analytes were separated using an Agilent Technologies InfinityLab Poroshell 120 EC-C18 column (50 mm × 2.1 mm, particle size 2.7 μm, part no. 699775-902) maintained at 50 °C. The mass spectrometer was calibrated using Pierce Triple Quadrupole Calibration Solution Extended Mass Range solution. For MS/MS acquisition: source fragmentation 0 V, collision energy 30 V, CID gas 1.5 mTorr (argon).

Positive mode analysis was performed with an atmospheric pressure chemical ionization (APCI) ion source. Mobile phase A was 99.9% water with 0.1% formic acid (v/v). Mobile phase B was 99.9% acetonitrile with 0.1% formic acid. The A/B gradient started at 20% B for 1 min after injection and increased linearly to 100% B at 5 min, held at 100% B for 10 min, using a flow rate 0.6 ml min⁻¹. Full Scan Q1 mass spectrometer parameters were as follows: spray current, static; positive ion discharge current, 4; negative ion discharge current, 10; ion transfer tube temperature, 275 °C; vaporizer temperature 35 °C; sheath, auxiliary and spare gas 45, 5 and 1, respectively. Samples were analysed with an m/z range of 200–1,000.

Negative mode analysis was performed with a HESI ion source. Mobile phase A was 94.9% water, 5% methanol and 0.1% formic acid (v/v) containing 10 mM ammonium acetate. Mobile phase B was 99.9% methanol with 0.1% formic acid. A/B gradient started at 15% B for 1 min after injection, increased linearly to 100% B at 20 min and was held at 100% B for 4 min, using a flow rate 0.6 ml min⁻¹. Full Scan Q1 mass spectrometer parameters were: spray voltage, 2.5 kV in positive mode; ion transfer tube temperature, 350 °C; vaporizer temperature, 350 °C; sheath, auxiliary and spare gas 60, 15 and 2, respectively. Samples were analysed with an m/z range of 200–1,000.

Chol^Alk-Sulf in hepatic portal vein blood was monitored with selective reaction monitoring with mass transitions: m/z 465.3→96.94 and collision energy at 30 V.

RT–qPCR analysis of Bt_0412 and Bt_0416

B. thetaiotaomicron (Bt) was maintained in BHIS media. One millilitre of stationary phase Bt was diluted in 25 ml of 10% tryptone medium and cultured as described in Bjursell et al.⁵¹. After 6 h, the log phase culture was diluted again (1 ml in 25 ml) in fresh tryptone medium containing either vehicle, lactose, cholesterol or lactose and cholesterol. For cholesterol-containing experiments, cholesterol was added to a final concentration of 10 μM. The cultures were harvested after 3 h incubation via centrifugation at 4,000g for 20 min. After removing the supernatant, bacterial pellets were resuspended in 125 μl of PBS and 150 μl of TRIzol reagent (Thermo Fisher Scientific), along with 0.5 mm zirconium beads, homogenized on a Mini-BeadBeater for 1 min and then moved to ice for 3 min. Thirty microlitres of chloroform was added and lightly inverted. Samples were centrifuged at 18,000g at 4 °C for 20 min. Then 300 μl of the RNA-containing fraction was applied to the RNA Clean & Concentrator-5 (Zymo Research) kit and RNA purified as per the manufacturer’s protocol. Some 20 μg of RNA was then reverse transcribed via the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as per the manufacturer’s protocol utilizing the random primers supplied with the kit. RT–qPCR was carried out using the complementary DNA to Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) with the qPCR primers listed in Supplementary Table 1 on a QuantStudio 7 Real-time PCR system (Applied Biosystems). All transcript quantifications were normalized to the 16S rRNA gene quantification.

Bacterial growth assay in cholesterol-supplemented media

Bacteria were maintained in BHIS broth. Cultures were allowed to reach stationary phase in BHIS and subsequently incubated at 22 °C for 24 h before use. Cholesterol minimal media was prepared by adding 1% Tween 80 (v/v) to the media and adding cholesterol to saturation. Excess cholesterol was removed via centrifugation, and a cholesterol dilution series was established. Cholesterol concentrations were determined via LC–MS. Input bacteria was normalized, inoculated into the various media supplemented with cholesterol, and cultured at 37 °C in an anaerobic chamber in a 96-well plate. The OD₆₀₀ values of the bacterial cultures were read using a BioTek Epoch 2 microplate spectrophotometer at 6 and 30 h.

Evaluation of transfer of labelled bacterial Chol^Sulf to mouse hepatic portal vein

To specifically track the transfer of bacterial Chol^Sulf, 1 ml of BtΔtdk (cholesterol sulfotransferase competent) or BtΔtdkΔ0416 (cholesterol sulfotransferase null) was inoculated into 25 ml of prewarmed (37 °C) 25 μM Chol^Alk supplemented minimal media^86,93 consisting of 13.6 g of KH₂PO₄, 0.875 g of NaCl, 1.125 g of (NH₄)₂SO₄, 5 g of glucose, (pH to 7.2 with concentrated NaOH), 1 ml of hemin solution (500 mg dissolved in 10 ml of 1 M NaOH, then diluted to final volume of 500 ml with water), 1 ml of MgCl₂ (0.1 M in water), 1 ml of FeSO₄⋅7H₂O (1 mg per 10 ml of water), 1 ml of vitamin K3 (1 mg ml⁻¹ in absolute ethanol), 1 ml of CaCl₂ (0.8% w/v), 250 μl of vitamin B12 solution (0.02 mg ml⁻¹) and 5 g of l-cysteine hydrochloride anhydrous). After incubation overnight at 37 °C, bacterial pellets were harvested by centrifugation at 4,000g for 20 min at room temperature. The cells were pelleted and washed twice with filtered sterile 0.1% BSA/ PBS to remove residual Chol^Alk and the resulting bacterial cells were resuspended in sterile PBS. The gavage amount of BtΔtdk(Chol^Alk) or BtΔtdkΔ0416(Chol^Alk) was adjusted to an OD₆₀₀ of ~1.0 per 200 μl of PBS. One millilitre of BtΔtdk(Chol^Alk) and BtΔtdkΔ0416(Chol^Alk) were frozen by liquid nitrogen and dried via lyophilization for verifying the presence of Chol^Alk-Sulf.

After 4 d of acclimation in a climate-controlled environment with 12 h light and dark cycles, nine 6-week-old Swiss Webster mice (Taconic Biosciences) were randomly assigned to two groups and orally gavaged once with either BtΔtdk(Chol^Alk) or BtΔtdkΔ0416(Chol^Alk). Mice were reared on a standard sterilized breeder diet (LabDiet 5021) with ad libitum access to autoclaved water. After 2 h, mice were euthanized by CO₂ followed by cervical dislocation. Some 30 IU ml⁻¹ of heparin was added to the body cavity around the hepatic portal vein before collecting the blood samples by Pasteur pipette⁸⁶. Hepatic portal vein blood was then frozen by liquid nitrogen and dried via lyophilization for metabolome extraction.

Human stool metabolomics

Human stool samples for testing the ability of human gut microbes to produce Chol^Sulf were analysed as subset of a study measuring stool metabolites during the first 3 months of life. The study protocol was approved by the Cornell Institutional Review Board for Human Subjects Research (protocol no. 2007009697) and all participants provided written informed consent. For infant participants, consent was provided by their immediate caregiver. Study incentives included a 3-month supply of nappies. Two stool samples collected from infant diapers (male, 0–3 months of age) were stored at −20 °C until thawed for use in ex vivo cultures as described above (Ex vivo culture of live faecal microbes with cholesterol or Chol^Alk).

Statistical analysis

No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications^55,93. Statistical tests were performed using GraphPad Prism 9 or in R. No animals or data points were excluded from the analysis. Data distribution was assumed to be normal but this was not formally tested. Owing to the nature of the treatment conditions, data collection and analysis were not performed blind to the conditions of the experiments. Statistical tests are denoted in the figure legends and Methods.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.