Generation of sterile spent culture broth filtrates
All tested bacteria were from the human gut microbial library constructed by Quantihealth Co. Ltd, Beijing, China (https://microbe.quantibio.com/). More specifically, all strains were isolated from the mixture of fecal samples donated by healthy adults in Hainan province, China. Each strain was purely cultivated, identified by mass spectrometry, named with a unique code, and then cryopreserved at -80 °C refrigerator.
For screening assay, each bacterial clone was pin transferred from the cryopreserved tube onto a solid YCFA medium (Solarbio) plate and was grown for 24-48 h at 37 °C under anaerobic conditions. A single colony was inoculated into liquid YCFA medium and cultured at 37 °C for 48 h under anaerobic conditions to obtain the first generation (F1) bacterial solution. 10% (v/v) of F1 bacterial solution was inoculated into fresh liquid YCFA medium and cultured at the same conditions for 48 h to generate F2 solution. Following the similar step as F2 production, the working bacterial solution F3 was further obtained from 10% (v/v) F2. After centrifugation at 1,600 × g for 15 min, the supernatant part was collected and then filtered using a Millipore filter (0.22 μm). These sterile spent culture broth filtrates were used as screening samples in the study.
Cellular lipid accumulation assay
HepG2 cells were purchased from ATCC and were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) at 37 °C with 5% CO2. For lipid accumulation assay, HepG2 cells were seeded in 96-well plates containing 100 μl DMEM. When the confluence reached 85%, the medium was replaced with 70 μl fresh DMEM supplemented with 100 μM oleic acid (OA, Sigma) and 30 μl spent culture broth filtrate or YCFA medium. After incubation for 22-24 h, lipid accumulation was evaluated by oil red O staining or TG quantification kit (Solarbio) as described previously . Each experiment (n = 8 for oil red O staining, n = 4 for TG determination) was repeated in triplicate. Liquid YCFA medium and fenofibrate (10 μM, Sigma) were used as the negative and positive control, respectively. Specifically, (1) for the large-scale bacteria screening, HepG2 cells were first stained with oil red O solution at room temperature for 30 min, and then DMSO was added to dissolve stains attached to cells, followed by measuring OD value at 358nm in a microplate reader. Lipid-lowering rate of each examined bacterial strain was evaluated and ranked according to the calculation of [(YCFA OD358 - Sample OD358) / YCFA OD358]*100%. (2) To confirm the lipid-lowering efficacy of Blautia producta, BODIPY staining and intracellular TG content were measured to corroborate the screening result according to the manufacturers’ instructions.
All the animal experiments were performed in accordance with the National Institutes of Health regulations for the care and use of animals in research. The protocol was approved by the medical ethics committee of Peking Union Medical College (Nos. YZS201904021; YZS201910013; YZS202105022). All efforts were made to minimize animal suffering.
(1) To assess the anti-hyperlipidemic effect of live Bl. producta, 24 male C57BL/6J mice (8-week-old, 20-24 g, Vital River Laboratory Animal Technology) were divided into three groups with eight animals in each group. One group was used as blank control and continued to feed on normal chow (Chow group) while other groups were fed HFD (60% kcal fat as indicated, Beijing HuaFuKang Bioscience). Animals on HFD were gavaged with Bl. producta (HFD+Bl. producta group, 109 CFU per animal per day) or an equal volume of YCFA medium (HFD group). Bodyweight was assessed weekly. After 4 weeks of treatment, mice were fasted overnight, anesthetized in chambers saturated with isoflurane, then sacrificed by cardiac puncture. Stools for metagenomic analysis were collected on the day before animals were euthanized. Blood was drawn in 1.5 ml centrifuge tubes, and sera were separated for estimation of serum levels of total triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and high-density lipoprotein cholesterol (HDL-c) by respective kits (Nanjing jiancheng Bioengineering Institute). Liver samples were weighed and snap-frozen in liquid nitrogen for sequential biochemical analysis.
(2) To assess the dynamic impact of live Bl. producta on gut microbiota in mice, 16 male C57BL/6J mice (8-week-old, 20-24 g) were randomly divided into two groups, fed on HFD, and gavaged with Blautia producta (HFD+Bl. producta group, 109 CFU per animal per day) or an equal volume of YCFA medium (HFD group) for 8 weeks. The fecal materials were obtained from each mouse on Days 1, 3, 7, 14 and 28 after treatment. At the end of the experiment, mice were fasted overnight, anesthetized with isoflurane, and sacrificed by cardiac puncture. Blood was drawn in 1.5 ml centrifuge tubes, and sera were separated for parameters evaluation. Liver samples were weighed and snap-frozen in liquid nitrogen for sequential biochemical analysis or fixed in 4% paraformaldehyde (Solarbio) for histological analysis.
(3) To assess the anti-hyperlipidemic effect of 12-methylmyristic acid (12-MMA), 16 male C57BL/6J mice (8-week-old, 20-24 g) were randomly divided into two groups and fed HFD. The test group (HFD+12-MMA) was gavaged with 12-MMA (40 mg/kg, Sigma), while the HFD group was given an equal volume of solvent (1% tween 80 + 0.5% carboxymethyl cellulose sodium (CMC-Na, Sigma)) for four weeks. At the end of the experiment, mice were fasted overnight, anesthetized in chambers saturated with isoflurane and then sacrificed by cardiac puncture. Blood was drawn in 1.5 ml centrifuge tubes, and sera were separated for parameters evaluation. Liver and fat samples were weighed and snap-frozen in liquid nitrogen for sequential biochemical analysis or fixed in 4% paraformaldehyde (Solarbio) for histological analysis.
Histology and immunohistochemistry analysis
The liver and fat tissues of each mouse were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into slides with a thickness of 4 μm. Liver and fat tissue sections were stained with hematoxylin and eosin (H&E) for histological analysis. For oil red O staining, liver tissues from the same liver lobe were cut into small pieces, then the frozen sections were rinsed in distilled water and stained with 0.2% oil red O (Sigma) and 60% 2-propanol (Sigma) for 10 min at 37 oC. For immunohistochemistry analysis, fat slides were rinsed in 0.01 mol/L sodium citrate (pH 6.0) and heated for 20 min in a microwave to retrieve antigen. The sections were blocked in blocking buffer containing 5% goat serum, 2% BSA, 0.1% Triton X-100 and 0.1% sodium azide in PBS, then incubated overnight with anti-UCP1 (Cell Signaling) by a dilution of 1:100 at 4oC. After being washed twice in PBS, slices were incubated with secondary antibodies (Cell Signaling) for 1 h at room temperature. Slides were counterstained with H&E. All digital pictures were acquired using an EVOS X1 microscopy (Thermo Fisher Scientific).
The microbial genomic DNA of fecal samples were extracted by DNeasy PowerSoil kit (QIAGEN) and subjected to 1% agarose gel electrophoresis for evaluation. Concentration and purity of microbial DNA were determined with NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific) and Qubit 3.0 fluorometer (Thermo Fisher Scientific).
The gut microbial composition was determined by shotgun sequencing of the fecal samples collected from each mouse. Libraries were prepared using KAPA HyperPlus Library Preparation kit (KAPA Biosystems) and quantified by KAPA Library Quantification Kits (KAPA Biosystems) following the manufacturer’s instructions. Shotgun sequencing was performed on Illumina NovaSeq 6000 System (Illumina) at a depth of 1Gb. Cluster generation, template hybridization, isothermal amplification, linearization, blocking, denaturing and hybridization of the sequencing primers were performed according to the workflow indicated by Illumina.
As previously described , low-quality reads were removed from the raw data by using MOCAT2 Sequencing adapters were removed by using Cutadapt software (version v1.14,-m 30). Then SolexaQA package was used to remove the reads with a threshold of less than 20 or the length of less than 30bp. The reads which could be aligned with the mouse genome (Mus musculus, GRCm38) were cleaned by using SOAP aligner software (v2.21, -M 4 -l 30 -v 10). The clean reads were assembled by SOAP de novo software (an iterative De-Bruijn Graph De Novo Assembler) using the parameters of -d 1,-M 3,-R ,-u,-F to get the scaftigs of at least 500bp. Genes were predicted using MetaGeneMark. A non-redundant gene catalogue was constructed with CD-HIT using the parameters of c 0.95 –aS 0.9. The clean reads were mapped onto the gene catalogue with the length of at least 100bp using BWA software to calculate the gene abundance.
Sequencing data analysis
α-Diversity was calculated by a vegan (2.5-6) package and presented by Shannon and Simpson indices. The principal coordinate analysis (PCoA) and nonmetric multidimensional scaling (NMDS) were calculated based on the Bray-Curtis distance using vegan (2.5-6) package. Microbial community composition was analyzed using Metaphlan2 software. Briefly, the query reads were mapped against the reference genomes in RefSeq database (version 82) with a 97% identity threshold. The linear discriminant analysis (LDA) effect size (LEfSe) method (https://github.com/biobakery/lefse) was used to identify species that show statistically significant differential abundances among groups. Heat maps were generated by using the R package “heatmap”.
Twelve reference genomes of Bl. producta were used to perform pan-genomic analysis, in which seven genomes were downloaded from NCBI RefSe or GenBank database and five genomes were constructed by de novo sequencing of pure Bl. producta cultures. Genes annotation was obtained using prokka software (https://github.com/tseemann/prokka). The pan-genomic analysis was performed by ppanggolin software (https://github.com/labgem/PPanGGOLiN) to acquire gene matrix, jaccard distance-based UPGMA clustering tree, and KEGG functional annotation.
The untargeted metabolomics profiling of the pure microbial culture was performed on the metabolomic platform of Shanghai Biotree biomedical technology Co. Ltd.
(1) Sample preparation: The frozen samples were kept on the dry ice-ethanol bath. About 50 mg material was accurately weighed in an Eppendorf Safelock microcentrifuge tube, to which 25 mg of pre-chilled zirconium oxide beads, 10 μl of internal standard, and 50 μl of 50% pre-chilled methanol were added for automated homogenization (BB24, Next Advance, Inc.). After centrifugation at 14,000 g and 4oC for 20 min (Microfuge 20R, Beckman Coulter, Inc., Indianapolis, IN, USA), the supernatant was carefully transferred to an autosampler vial (Agilent Technologies, Foster City, CA, USA). Each aliquot of 175 μl of pre-chilled methanol/chloroform (v/v=3/1) was added to the residue for the second extraction. After centrifugation at 14,000 g and 4oC for 20 min, the supernatant was carefully transferred to an autosampler vial. All samples in autosampler vials were evaporated briefly to remove chloroform using a CentriVap vacuum concentrator (Labconco) and further lyophilized with a FreeZone freeze dryer equipped with a stopping tray dryer (Labconco).
(2) Chromatographic separation: Chromatographic separation was performed on the Agilent 1200 Series HPLC instrument (Agilent) combined with an ACE Excel 3 C18 column (100 mm × 2.1 mm, 3.0 μm), an autosampler, quaternary pump, and vacuum degasser. The separation conditions were as follows: flow rate of 350 μl/min, column temperature of 35 °C, injection volume of 3 μl, sampler tray of 4 °C, mobile phase A (water + 0.1% formic acid), and mobile phase B (acetonitrile). The gradient elution programs were performed as described below: 0–1.5 min, 2% B; 1.5–4.0 min, 2–20% B; 4.0–9.0 min, 20–60% B; 9.0–18.0 min, 60–98% B; 18.0–19.0 min, 98% B; 19.0–19.5 min, 98–2% B; and 19.5–21.5 min, 2% B in the positive ion mode; 0–1.5 min, 2% B; 1.5–3.0 min, 2–25% B; 3.0–7.0 min, 25–45% B; 7.0–8.0 min, 45–60% B; 8.0–15.0 min, 60–98% B; 15.0–16.0 min, 98% B; 16.0–17.5 min, 98–2% B; and 17.5–19.0 min, 2% B in the negative ion mode.
MS was performed on the Agilent 6530 Q-TOF mass spectrometer (Agilent) coupled with electrospray ionization (ESI) source with detection capability for both negative and positive ion modes. MassHunter Workstation software (Agilent) was employed for the system operation. The operation conditions of mass spectrometer were as follows: capillary temperature of 320°C, capillary voltage of 4,000 V for positive ion mode and 3,500 V for negative ion mode; nebulizer of 35 psig; sheath gas flow rate of 12 L/min; collision energy of 35 eV; drying gas temperature of 300 °C and drying gas flow rate of 6 L/min. The mass range was set from 50 to 1100 Da with full scan mode. For the QC samples, pooled QC samples of data were acquired to supervise the real samples’ variability in the overall analytic run.
Oral glucose tolerance test (OGTT)
As previously described , before the OGTT test, mice were fasted for 6 h and then gavaged of 2 g/kg glucose. The blood glucose concentration in the tail vein was monitored at 0, 30, 60, 90 and 120 min after glucose administration (p.o.) using a glucose meter (Roche).
Quantitative real-time quantitative PCR (qRT-PCR) assay
The inguinal fat tissue was used to evaluate the mRNA levels of key genes involved in fat browning. Total RNA extraction, first-chain cDNA synthesis, and quantitative PCR assays were performed as previously reported . The primers used for each gene were listed in Additional file 2: Table S7.
Fluorescence-based GPR120 activation assay
To evaluate the activating effect of 12-MMA on GPR120, we set up a fluorescence-based GPR120 activation assay using STC-1 intestine endocrine cells (purchased from ATCC) stably transfected with a yellow fluorescent protein-based cAMP indicator Flamindo2 , as well as a detection test of GLP-1 secretion. (1) For GPR120 activation assay, cells were incubated with different concentrations of 12-MMA or myristic acid (MA, Sigma) or dimethyl sulfoxide (as a vehicle) for 20 min after transient transfection of GPR120-expressing plasmid. The fluorescence signal intensity (FL) were recorded by Tecan Infinite M1000Pro Microplate Reader at excitation 485 nm and emission 535 nm. GPR120 activation was calculated as (FL12MMA or MA-FLDMSO)/FLDMSO*100%. (2) The secreted level of glucagon-like peptide-1 (GLP-1) in the culture medium was determined using mouse GLP-1 ELISA Kit (Solarbio) according to the manufacturer’s instructions.
Statistical analysis, graphing and figure assembly
Data are presented as means ± SEM. SPSS 17.0 and Prism 7 (Graphpad) were used for statistical analysis. The significance of group differences for normally distributed data was assessed by one-way ANOVA followed by Bonferroni post hoc tests. PERMANOVA (Permutational multivariate analysis of variance) was performed to evaluate the significance of group differences for PCoA and NMDS analysis. P* < 0.05 was considered statistically significant. All final figures were assembled using Adobe Illustrator.