Study subjects
58 subjects were recruited in this study from a cohort of Jixian, Tianjin. They were divided into two groups according to the global hypertension practice guidelines[21], 23 subjects in the control group with SBP<140 mmHg and DBP<90 mmHg, 35 subjects in the hypertension group with SBP ≥ 140 mmHg or DBP ≥ 90 mmHg, the blood pressure was measured in a sitting posture with medical electronic sphygmomanometer after 5 minutes rest, and the mid-arm was at heart level, it took 3 times with an interval of 1min and used the average data.
This study was approved by the ethical committee of Peking University Third Hospital, and the informed consent were obtained from all subjects before the study. The exclusion criteria of subjects were as follows: A history of drug treatment (such as antihypertensive drugs, lipid-lowering drugs, anticoagulants, etc.); Suffering from other cardiovascular diseases (such as coronary heart disease), diabetes and kidney diseases; Suffering from gastrointestinal diseases and cancers; Suffering from oral diseases (such as dental caries, etc.); A history of antibiotics or mouthwash treatment within three months; Suffering from respiratory infection within one month;
Tongue coating sample collection and DNA extraction
Tongue coating was collected using oral sterile swabs before breakfast, the tongue was scraped from the root of the tongue to the tip 10 times, the swab was snapped into DNA preservation solution and stored at -80 ℃ in 4h. Lysozyme (10 mg/mL, 100 μL/sample) was pretreated before DNA extraction, and DNA was extracted from each tongue coating sample following the protocol of QIAamp DNA Mini Kit (Qiagen, Germany).
Metagenomic sequencing and Library construction
The DNA libraries were constructed following the Illumina TruSeq DNA Sample Prep v2 Guide (Illumina, Inc., San Diego, CA, USA), and all samples were subjected to 150 bp paired-end sequencing on an Hiseq X-ten platform (Illumina, Inc., San Diego, CA, USA).
Quality control of Illumina reads
Illumina raw reads should be screened as following criteria: Removing reads containing adaptor contamination, more than three ambiguous N bases, low quality (Q<20) bases or less than 60% of high-quality bases; Abandoning the host genomes and selecting the bacterial genomes with SOAPaligner (version 2.21)[22] for further analysis.
Microbial relative abundance profiling
Bacterial data were aligned to the NCBI database (National Center for Biological Information, http://www.ncbi.nlm.nih.gov) for detection of known microbiota by SOAPaligner 2.21[22], and then classified as Kindom, Phylum, Class, Order, Family, Genus, Species to count classification and taxonomic relative abundance, the total abundance of each species in a single sample is 1.
Genome assembly, gene prediction and gene catalog construction
The assembly of reads was carried out with a series of k-mer (51, 55, 59, 63) by SOAPdenovo (version 2.04)[23], and contigs longer than 500bp were kept for further analysis. Software MetaGeneMark[24] was used to predict open reading frames (ORFs) not less than 100bp. CD-HIT (version 4.5.7) [25] was used for pairwise comparison of predicted ORFs which were used for construction of a non-redundant gene catalog set[26].
Gene functional annotation and functional profiling
BLAST (Version 2.2.28+) was used to align the non-redundant gene catalog set with KEGG (Kyoto Encyclopedia of Genes and Genomes) and eggNOG (evolutionary genealogy of genes) database for the annotation information. And then the relative abundance of all orthologous genes was accumulated to generate the relative abundance of each KO/NOG.
Quantitative Real-time PCR
A. odontolyticus were grown at 37 °C in 3mL BHI broth supplemented with sanguinarine (10 μg/mL) until the OD570 reached 0.15. Total RNAs were extracted by RNAprep pure Cell / Bacteria Kit (DP430, Tiangen, China) according to the manufacturer’s protocol. 1 μg total RNAs was used in 20 μL reaction volumes to generate cDNA by using a FastKing RT kit (KR116, Tiangen, China). QRT-PCR was performed using a Talent qPCR PreMix (SYBR Green) kit (FP209, Tiangen, China) with forward primer (5′-CGAAGTACTGCTGCGCCATC-3′) and reverse primer (5′- TCAACGTTGGCCTCGTCTAC-3′) primers for the AO-13 related MFS gene, forward primer (5′-CAGACCGCCATGACGATGAT-3′) and reverse primer (5′-ACGTATGCCGATGTCGATGG-3′) for the AO-19 related MFS gene. The internal control is forward primer (5'-CTTTGGGATAACGCCGGGAAAC-3') and reverse primer (5'-CTACCCGTCAAAGCCTTGGT-3'). The data analysis was done through 2-∆∆Ct method.
Bacterial strains
This study contained two strains, A. baumannii (JCM 6841) was purchased from China General Microbiological Culture Collection Center (CGMCC) which was grown in LB broth at 37°C, A. odontolyticus (ATCC 17929) was purchased from American Type Culture Collection (ATCC) which was grown in BHI broth at 37°C.
Bioinformatics Analysis
According to the five common bacteria of tongue coating, including Actinomyces, Streptococcus, Veillonella, Neisseria, and Prevotella, and ten differential strains with high relative abundance respectively enriched in two groups, all complete bacterial genomic DNA sequences was downloaded from eHOMD. If the strain had several complete genomes, the largest genome was selected for G4 analysis. To further analyze the G4 sequence of A. baumannii and A. odontolyticus which were considered as hypertension-associated bacteria, 244 complete genomes of A. baumannii and 2 genomes of A. odontolyticus were downloaded from National Center for Biotechnology Information.
Putative G4 sequences were predicted by G4RNA screener[27] (http://gitlabs cottgroup.med.usherbrooke.ca/J-Michel/g4rna_screener). The sliding window of 30 nucleotides (nt) moving with steps of 15 nt was used to search potential G4s. The thresholds were as follows: cGcC score >4.5, G4Hunter score >0.9, G4NN score > 0.5. Putative G4 sequences that met the scoring threshold and had gene annotation function remained.
Circular dichroism and thermal stability studies
CD spectroscopy was recorded on a J-815 CD spectrometer (JASCO, Japan) at 20°C, over 220-330 nm with the scanning speed at 100 nm/min. Each sample was measured three times. DNAs with 5nM final concentrations were prepared in 30mM Tris-HCl and 0-150 mM KCl. The mixtures were then heated at 95°C for 15 min and gradually cooled to room temperature. For CD thermal stability studies, spectra were recorded with a temperature range from 20 to 95 °C, and the temperature rising rate was 3°C/min.
ESI mass spectrometry (ESI-MS)
The ESI-MS experiments were performed on Bruker SolariX-XR mass spectrometer (Bruker, Billerica, MA, USA) with an ESI source. All samples were tested in a negative ion mode, with a capillary voltage of 2.7 kV, and the samples were injected with the flow rate at 120 μL/h. To test the binding properties, the samples were prepared in the solutions with 25% CH3OH to have better efficiency, the sanguinarine and DNA sequences were at 4:1 ratio.
Bacterial growth assays
To monitor the effect of sanguinarine on A. odontolyticus and A. baumannii, the bacterial suspension containing agents (5, 10, 20 µg/mL sanguinarine) was grown under the anoxic condition at 37℃, the control was free of sanguinarine. At designated time points (0, 2, 4, 6,10,12,14 h), the cell broths were measured the OD with a microplate reader at 570 nm.
Biofilm formation assay
A. odontolyticus (106 CFU/mL) was inoculated in culture dishes with transparent bottom to form biofilms, containing sanguinarine (5 µg/mL, 10 µg/mL or 20 µg/mL) at 37℃ and in anaerobic condition. The control was free of sanguinarine. After 24h culture, the dishes were washed two times with PBS. Subsequently, 1.5 μL SYTO 9 dye from LIVE/DEAD BacLight Bacterial Viability Kit were diluted in 1mL PBS and then the diluent was added into each dish. All wells were incubated for 30 min in the dark. After the staining, the wells were washed twice with PBS and each well was added 1mL 4% paraformaldehyde for 15 min for fixation. The images were visualized by laser confocal microscope (Carl Zeiss, LSM-780, Germany) at wavelengths of 480 nm excitation and 500 nm emission.
Assessment of nitrite concentration
The overnight cultured A. odontolyticus (OD570 of 0.15) was diluted into BHI broth (1:100 dilutions) supplemented with 20 μg/mL, 10 μg/mL, 5 μg/mL, 2.5 μg/mL, 1.25 μg/mL sanguinarine, meanwhile, KNO3 was added at the final concentration of 1 mM as the substrate. After 24h under anoxic condition, the bacterial solution was vortexed and centrifuged at 12,000 rpm for 5 min to collect the supernatant. Using a nitrite colorimetric assay kit (Elabscience, E-BC-K070-S, China), supernatant nitrite levels were determined. The absorbance was read at 550 nm.
LC-MS/MS for quantitation of TMA
Overnight cultured A. odontolyticus (OD570 of 0.15) was diluted into BHI broth (1:100 dilutions) supplemented with 10 μg/mL sanguinarine and 10-4 mM γ-Butyrobetaine at 37°C under anoxic condition for 24h. Samples (20 µL bacterial suspension) were mixed with 80 µL of 10 µM d9-TMA in methanol to precipitate protein. LC–MS/MS analysis was conducted on a Q TRAP5500 mass spectrometer. The following settings were selected: curtain gas, 20 psi; source temperature, 600 °C; gas 1 and gas 2, 35 and 50 psi; spray voltage, 4.5 ESI+ kV; collision activation parameter, medium. Supernatants (10 µl) were analyzed by injection onto a silica column (Luna 5u Silica 100A, 2.0*150 mm) at a flow rate of 0.5 mL/min. The temperature was set at 35°C. Mobile phases A consisted of 1‰Propionic Acid in LC–MS grade water and Mobile phases B consisted of 1‰FA in MeOH. And the Gradient (B %) was 2% for 1 min, 95% for 11 min, 2% 6.5 min, and stop at 7 min. The internal standard d9-TMA was used for quantification.
Biolog ECO microplates assay
A. odontolyticus were prepared in BHI solid broth for biolog assays. Monoclonal colonies were mixed in IF-A liquid until reaching 90-98% turbidity. Then the IF-A liquid (150 μL) was added to ECO microplates, which were cultured at 37°C in the anaerobic condition. After 48h, the ECO microplates were read at absorption 590 nm using a BIOLOG microplate reader (MOLECULAR DEVICES, United States). And AWCD was used to test the carbon source utilization capability of A. odontolyticus[28].
Statistics.
R software was used to perform all statistical analyses in metagenomics. Wilcox rank-sum test was used to calculate the significance of different taxonomic (phylum, class, order, family, genus, species). Other data were expressed as mean ± SEM. Statistical analysis between two groups used the Student’s t-test. Statistical analysis was performed on SPSS 23.0.