Antibodies
The primary antibodies include mouse monoclonal anti-GPX4 (1:3000, Cat No. 67763-1-Ig), rabbit polyclonal anti-SLC7A11 (1:1000, Cat No. 26864-1-AP), rabbit polyclonal anti-SLC3A2 (1:20000, Cat No. 15193-1-AP), mouse monoclonal anti-NF-κB p65 (1:1000, Cat No. 66535-1-Ig), rabbit polyclonal anti-ASCL4 (1:6000, Cat No. 22401-1-AP), rabbit polyclonal anti-COX2 (1:2000, Cat No. 12375-1-AP), rabbit polyclonal anti-LOX (1:600, Cat No. 17958-1-AP)(Proteintech Group Inc., Rosemont, IL, USA), goat anti-rabbit IgG (1:50000, H + L, Cat No. RGAR001), and goat anti-mouse IgG (1:20000, H + L, Cat No. RGAM001) were purchased from Proteintech Group, Inc (Rosemont, IL, USA),
Strains and cells and their culture
Streptococcus strain D19T was collected from the oropharynx of healthy children and cultured in bacto brain heart infusion medium(Solarbio, Beijing, China) at 37℃, 180r/min, for 18hours. Acinetobacter baumannii S2009-4 originates from the Laboratory of Shenyang Medical University Affiliated Central Hospital and is cultured in broth medium(Solarbio, Beijing, China) under conditions at 37 ℃, 180 r/min, for 18 hours.
Human bronchial epithelium BEAS-2B cells and 16HBE cells were purchased from Beijing Dingguo Changsheng Biotechnology Co., LTD. BEAS-2B and 16HBE cells were cultured in DMEM(Hyclone, Logan, UT, USA) medium containing 10% fetal bovine serum(Hyclone, Logan, UT, USA), 100 units/mL penicillin(Genview, Australia), and 100 units/mL streptomycin solution(Genview, Australia) at 37℃ and 5% CO2.
Antibacterial assay
The concentration of Acinetobacter baumannii bacterial solution in the logarithmic phase was adjusted to 1×106 CFU/mL, and 100µL was removed and evenly spread on nutrient AGAR plates. Using the disk diffusion method, 200µL of the sample solution to be tested was added to the disk and placed in a bacterial incubator for 18 h at 37°C. The diameter of inhibition was recorded with a vernier caliper.
Fermentation broth conditions determination
D19T single colonies were inoculated into brain heart infusion medium and incubated at 37°C, 180 r/min, for 18h. The inhibitory diameter was measured under different fermentation conditions with different pH (3–11), fermentation temperature (28–43℃) and fermentation time (12-60h).
Antibacterial components determination
Antibacterial component determination: D19T fermentation broth was centrifuged at 4 ℃ and 12000 r/min for 10 minutes to remove bacterial cells and obtain the supernatant of the fermentation broth. Add (NH4) 2SO4 solution to the supernatant to a saturation of 30%, and precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12000 r/min for 10 minutes. The protein precipitate was dissolved in 50 mmol/L phosphate buffer and desalinated using a dialysis bag(Yeasen Biotechnology, Shanghai, China). Measure the antibacterial effects of the precipitated protein and supernatant separately.
The determination of the optimal concentration of (NH4) 2SO4: The method for obtaining the supernatant of D19T fermentation broth is consistent with the method mentioned earlier. Add (NH4) 2SO4 solution to the supernatant, and the saturation of (NH4) 2SO4 in the final supernatant ranges from 20–80%. Precipitate overnight at 4 ℃. The next day, the fermentation broth was centrifuged at 12000 r/min for 10 minutes. The protein precipitate was dissolved in 50 mmol/L phosphate buffer and desalinated using a dialysis bag. Measure the antibacterial effect of proteins precipitated with different saturation levels (NH4) 2SO4 separately.
Antibacterial proteins Isolation and purification
The protein solution was loaded on a Sephadex G-15(Sigma, Louis, MO, USA) column and eluted with PBS solution at a flow rate of 1.5 mL/min. The elution peaks of each protein were detected and collected under ultraviolet light at 280nm to determine the inhibitory diameter. The bacteriostatic active fractions were collected and concentrated and then processed by cellulose DE-52 chromatography(Biosharp, Anhui, China). The flow rate was 1.5 mL/min and equilibrated in PBS solution until the baseline was stable. Linear gradient elution was carried out with 0 ~ 1.0 mol/L NaCL in PBS buffer, and the flow rate was controlled at 0.5 mL/min. The elution peaks of each protein were collected to determine the inhibitory diameter.
Minimum inhibitory concentration (MIC) determination
The antibacterial protein was diluted to 2, 4, 8, 16, 32 and 64 times with bacto brain heart infusion medium. 100µL protein solution was added to 96-well plate, and 10µL pathogen solution (1×106 CFU/mL) was added to each well. After mixing, the 96-well plate was cultured in a bacterial incubator at 37℃ for 24 hours. The minimum drug concentration without bacterial growth was read by the plate viable bacteria count method, which was the minimum inhibitory concentration (MIC) of the bacteria to the drug.
DNA and RNA exosmosis levels determination
DNA and RNA exosmosis levels of Acinetobacter baumannii were measured using Thremo Nanodrop 2000 detector(Thermo Fisher Scientific, Waltham, MA, USA). The operation procedure is summarized as follows: Run the Nanodrop when the sample measuring arm is off, add 2µL of distilled water to the optical fiber surface, lower the measuring arm, and set to zero. Add 1.0µL of the sample to be measured successively, and repeat the measurement for each sample to be measured 3 times.
Soluble protein levels determination
The level of soluble protein in Acinetobacter baumannii was determined by SDS-PAGE. The operation steps are summarized as follows: the suspension of Acinetobacter baumannii after treatment in the control group and the experimental group was centrifuge and the supernatant was removed to collect the bacteria. Acinetobacter baumannii in each group of samples were washed with pre-cooled PBS solution twice, the supernatant was removed by centrifuge and the bacteria were collected, then re-suspended in PBS solution and adjusted to the same bacterial density. The bacteria were treated in a metal bath at 100℃ for 10min, mixed upside down once every 2min, and the lysed cells released soluble proteins. Protein samples of 20µL were added for SDS-PAGE electrophoresis, dyed with Coomassie bright blue (Beyotime Biotechnology, Shanghai, China) for 40min, decolorized with distilled water, and photographed for analysis of protein expression.
Biofilm formation determination
100 µL Acinetobacter baumannii solution at a concentration of 1×106 CFU/mL was added to 96-well plate, and then antibacterial protein solution was added to the final concentration of 0, 1/2MIC and MIC, respectively, and cultured at 37℃ for 24 hours. The culture medium and non-adherent bacteria were washed with PBS buffer, methanol was fixed for 15min, the biofilm was stained with 2% crystal violet solution for 15min, and the cells were decolorized with 33% glacial acetic acid. The absorbance was determined at 630 nm. The broth medium without bacteria was used as a negative control.
Adhesion ability determination
500 µL of BEAS-2B and 16-HBE cells at a concentration of 5×104 cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37℃ and 5%CO2 overnight. The cells were first cultured in serum-containing DMEM for 3 days, and then starved in serum-free DMEM culture for 12h. After washing with PBS, 1mL of Acinetobacter Baumannii suspensions with a concentration of 1×108CFU/mL were added to each well, and 1mL of DMEM culture solution was added, mixed evenly, and incubated together for 2h. The cells were washed with PBS and fixed with 4% paraformaldehyde for 30min. The number of Acinetobacter baumannii adherens on the cell surface was observed and counted under the microscope. Non-adhesive bacteria (≤ 40 bacteria), adhesive bacteria (41–100 bacteria), strongly adhesive bacteria (> 100 bacteria).
Cell iron death determination
500 µL of BEAS-2B and 16-HBE cells at a concentration of 5×104 cells /mL were seeded onto cell slides in 6-well plates and incubated in at 37℃ and 5%CO2 overnight. Add 500µL DMEM containing ammonium ferric sulfate (II) into each well (the final concentration of ammonium ferric sulfate (II) is 100 mol/L), and incubate in the incubator for 30 minutes. After washing the cells with PBS, the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, and then treated with 1% Triton X-100 for 20 minutes. The cells were washed with PBS and incubated in an incubator with 500µL FerroGreen solution for 30 minutes. The intensity of each fluorescence signal was detected by GFP filter and BF filter in fluorescence microscope.
Inflammatory cytokines detection
The supernatant was obtained by centrifugation at 1000g for 20 minutes. Standard and sample holes were set up according to TNF-α, IL-6 and IL-8 instructions(Mibio, Shanghai, China). Add 50µL of standard product with different concentration to the standard product hole, and add 50µL of sample to the sample hole. Horseradish peroxidase (HRP) labeled detection antibody 100µL was added to each well and incubated in an incubator at 37℃ for 60min. Add 300µL washing solution to each well and repeat washing for 5 times. Add 50µL of substrate A and substrate B to each well and incubate at 37℃ for 15min without light. The absorbance of each hole was measured at 450nm wavelength by adding 50µL terminating solution to each hole.
Western Blot
Wash the cells once with a pre-cooled PBS solution to remove as much excess fluid as possible. 500µL RIPA lysate(Beyotime Biotechnology, Shanghai, China) containing 1mM PMSF(Beyotime Biotechnology, Shanghai, China) was added to the cells, and the entire lysate process was performed on ice. The protein samples were separated by SDS-PAGE gel electrophoresis at 80V, 20 min, 120V, 50 min. The protein on the gel was transferred to the PVDF membrane(Millipore, Boston, MA, USA) by wet transfer method at 300mA for 2 hours. The transferred PVDF film was washed with TBST and sealed overnight with 5% skim milk(Sigma, Louis, MO, USA) powder solution at 4℃. On the second day, they were incubated at room temperature for 1 hour with first antibody diluent and second antibody diluent, respectively. The protein was developed with enhanced ECL chemiluminescence reagent (Vazyme, Shanghai, China), and the gray values of the protein bands were calculated by Image J software.
Ferroptosis was detected by electron microscopy
Cells were collected and added to the fixative precooled at 4°C and placed at 4°C overnight. The fixative was decanted, rinsed with phosphate buffer, and samples were fixed with 1% osmic acid solution for 2h. "The samples were dehydrated with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, and 100%) and finally treated with acetone. The samples were embedded in the embedding agent and cut by a microtome. The sections were stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 10min, respectively, and observed under a transmission electron microscope.
Statistical analysis
Since each experiment was performed three times, the data is given as mean ± SD. A two-tailed Student 'st test was used to assess the differences between the two groups. Analysis of Variance (ANOVA) is used to assess differences between multiple sets of data. P value lower than 0.05 was considered significant. SPSS 19.0 was used to analyze the data.