Bacterial cultures, media and ethical considerations
All bacterial strains, culture conditions and sources used in this study are listed in Additional file 1: Table S1. RAW 264.7 cells were obtained from the College of Animal Medicine, Jilin Agricultural University Changchun, China. Luria-Bertani broth medium (LB) and Luria-Bertani agar medium (LBA) were purchased from Sangon Biotech (Shanghai, China), and Mueller-Hinton broth medium (MHB) was purchased from GL Biochem (Shanghai, China). Mice (weighing 16-20 g) were purchased from the Experimental Animal Center of Jilin University (Changchun-Jilin, China). All mice tests were carried out in accordance with the Regulations for Animal Experimentation at Jilin Agricultural University (JLAU08201409) and Laboratory Animal Care and Use Guidelines of National Institutes of Health (NIH Publication No. 8023).
Purification of the crude protein
According to the previously described methods of our laboratory [5], antibacterial protein CB6-C was purified through 100% ammonium sulfate precipitation, Sephadex G-75 column, QAE-Sephadex A 25 column and chromatography C4 column (5 μm, 4.6 mm × 250 mm, Agilent, USA). Briefly, purified B. velezensis CB6 was inoculated into 500 mL LB broth medium at 1% (v/v) and shaken at 37 °C for 48 h. The cultures were centrifuged at 4 °C and 8000 × g for 60 min, and then the supernatants were filtered twice through 0.22-μm membranes (BioLeaf, Shanghai, China) to remove cells. Next, ammonium sulfate was gently added to the collected supernatant to reach 100% saturation, stirred at 4 °C for 12 h, and allowed to stand overnight followed by centrifugation (12,000×g, 30 min, 4 °C). The sediment was resuspended in 10 mM phosphate-buffered saline (PBS, pH 7.4), and a 10 KD ultrafiltration tube was used to remove ammonium sulfate and small molecule compounds. The samples containing proteins larger than 10 KD were tested for antibacterial activity. The active samples were loaded onto a Sephadex G-75 (3 KD-70 KD, Sigma, USA) gel filtration column, connected to an AKTA purification system (Boston, USA) and eluted at 1 mL/min flow rate. The proteins were classified according to the different molecular weights, and the separated peaks were collected at 280 nm and tested for antibacterial activity. The collected antibacterial fraction was further purified by a QAE-Sephadex A 25 column. Different concentrations of NaCl (0, 0.1 M, 0.2 M and 0.3 M) in 100 mM Phosphate buffer (pH 7.0) were used to elute the antibacterial protein. Next, for further purification of the crude antibacterial proteins, active samples were applied to a reverse-phase high-performance liquid chromatography system (RP-HPLC, Agilent, CA, USA) equipped with a reverse-phase chromatography C4 column. Elution was performed by using a 5-90% linear gradient of acetonitrile containing 1% trifluoroacetic acid with 1 mL/min flow rate for 60 min. The separate peaks of the UV detector were recorded at 280 nm. For the above test, after each purification step, MRSA was used as indicator bacteria, and the agar diffusion method was used to determine the antibacterial activity [6].
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Molecular mass determination and LC-MS/MS analysis of antibacterial protein CB6-C
After purification of the protein using a C4 column, partially active samples were applied to a 12% polyacrylamide gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Mini-PROTEAN II Electrophoresis Cell, Bio-Rad Laboratories, USA) and subjected to a 100 V constant voltage for separation. Then, the gel was stained with Coomassie brilliant blue and washed three times with Phosphate buffer to remove impurities, and the molecular size of the purified antibacterial protein was determined. Subsequently, only a single band was cut and sent to gene sequencing company (Wuhan, China) to determine the protein sequence by using liquid chromatography-tandem mass spectrometry (LC-MS/MS, Bruker Daltonics, Germany).
Safety assays
The hemolytic activity of the antibacterial protein CB6-C was examined according to a protocol as described previously [7]. Briefly, the collected sheep blood cells were washed with PBS (pH 7.4) three times and diluted to 2% in PBS. Then, equal volumes of sheep blood cells were mixed with different concentrations of antibacterial protein CB6-C (0.5 µg/mL to 256 µg/mL) in tubes and incubated at 37 °C for 1 h. Subsequently, the mixtures were centrifuged at 1000 × g for 10 min, and the supernatants were transferred to 96-well plates. Equal amounts of PBS and 0.2% Triton X-100 were added as negative and positive controls, respectively. The absorbance of the mixtures was measured by the OD at 570 nm. The experiment was performed three times. RAW 264.7 cells were used to study the cytotoxicity of the antibacterial protein CB6-C. This test used the method described by Xu et al. [8]. In brief, RAW 264.7 cells were collected and washed twice with DMEM, and equal amounts of the cells were placed into 96-well plates at a density of 105 cells per milliliter, and cultured overnight at 37 °C under condition of 5% CO2. Then, equal amounts of antibacterial protein CB6-C were mixed with the cells, and the final concentrations of antibacterial protein CB6-C per well were 0.5 µg/mL to 128 µg/mL. After 16 h of culture at 37 °C, CCK-8 (10%, v/v) was added to each mixed cell’s well in the 96-well plates and cultured at 37 °C for 2 h. The absorbance was measured by the OD at 450 nm (microplate reader, TECAN GENios F129004, Tecan, Salzburg, Austria). The experiment was performed three times.
Antibacterial spectrum and minimum inhibitory concentration
The method described by Jia et al. [9] was adopted to determine the antibacterial spectrum and minimum inhibitory concentration (MIC) of antibacterial protein CB6-C. In short, the antibacterial protein CB6-C collected from RP-HPLC was condensed into powder. The powder was dissolved with PBS until reaching a concentration of 512 µg/mL and added to the first well in each row in 96-well plates, and multiple dilutions with PBS were performed at final concentrations ranging from 0.5 to 256 µg/mL. Concurrently, various indictor strains were inoculated into MHB broth medium and shaken at 37 °C and 180 rpm to cultivate to log phase growth (OD at 600 nm = 0.5). Then, the bacterial concentration was adjusted to approximately 105 CFU/mL using MHB medium, and an equal volume of bacteria was added to 96-well plates. After incubating at 37 °C for 18 h, the absorbance was measured by the OD at 600 nm (microplate reader, TECAN GENios F129004, Tecan, Salzburg, Austria). The MIC was defined as the lowest concentration with no growth of bacteria after incubation at 37 °C for 16-20 h.
Time-kill kinetics assay
The time-kill kinetics of MRSA were determined as previously described [10]. In brief, the MRSA cells at the logarithmic growth phase were adjusted to an OD of 0.5 at 600 nm, and the bacterial concentration of approximately 105 CFU/mL was obtained using MHB broth medium. Next, antibacterial protein CB6-C was added to a final concentrations of 1 × MIC, 2 × MIC and 4 × MIC, with an equal volume of PBS added to antibacterial protein CB6-C as a control followed by shaking at 37 °C and 180 rpm for 24 h. During this period, equivalent amounts of liquid were taken every four hours for serial tenfold dilutions, and then a 100 µl of the dilutions was spread on the LB plate, after which the colonies were counted. The experiment was performed three times.
Stability assays
The purified antibacterial protein CB6-C was used to test the temperature stability according to the method described by Tumbarski et al. [11]. Equivalent amounts of antibacterial protein CB6-C were put in the test tubes, sealed and then placed at 40 °C, 50 °C, 60 °C, 70 °C, 80 °C, 90 °C and 100 °C for 60 min and 121 °C (autoclaving for 15 min). To determine the effect of enzymes on the antibacterial activity of antibacterial protein CB6-C, a final concentration of 1 mg/mL of catalase (5000 µ/mg), pepsin (250 µ/mg), papain (800 µ/mg), trypsin (10,000 µ/mg) and proteinase-K (30 µ/mg) manufactured in Sigma-Aldrich, (Merck, USA) was added to equivalent amounts of antibacterial protein CB6-C samples. After incubation at 37 °C for 30 min, the enzyme reaction was stopped by heating at 100 °C for 10 min. To assess the influence of acid bases on antibacterial protein CB6-C activity, equivalent amounts of antibacterial protein CB6-C were adjusted to pH 2-12 by using HCl and NaOH and incubated at 37 °C for 1 h. Subsequently, all antibacterial protein CB6-C samples were readjusted to pH 7.0. To test the organic reagent effect on antibacterial protein CB6-C activity, 1% (v/v) methanol, isopropanol, Tween 20, Tween 80, acetonitrile, acetone and EDTA (Sigma-Aldrich, Merck) were incubated with antibacterial protein CB6-C at 37 °C for 1 h.
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For the experiment outlined above, untreated antibacterial protein CB6-C was used as a positive control, and the inhibition zones of the treated antibacterial protein CB6-C and positive control samples were measured to estimate the influencing factors of the antibacterial protein CB6-C. Each experiment was repeated three times.
Synergy with conventional antibiotics
The antibacterial effects of antibacterial protein CB6-C in combination with other antibiotics were evaluated by checkerboard tests [12]. In short, antibacterial protein CB6-C and antibiotics were prepared at final concentrations from 1 × MIC to 1/64 × MIC. Next, the same concentration of antibacterial protein CB6-C was added to the horizontal row of 96-well plates, and the same concentration of antibiotic was added to the longitudinal column of 96-well plates. Then, a 105 CFU/mL MRSA was added to each well and incubated at 37 °C for 10 h. Each test was performed three times. The fractional inhibitory concentration (FIC) index was calculated as follows:
where FIC ≤ 0.5 denoted synergy and 0.5 < FIC ≤ 1.0 denoted an additive effect.
Effect of metal ion on CB6-C activity
Effects of metal ions (K+, Co2+, Ni+, Mg2+, Mn2+, Ca2+, Ba2+, Fe3+ and Cu2+) on CB6-C antibacterial activity were carried out according to the protocol described previously [13]. Briefly, a 512 ug/mL of CB6-C was continuously twofold diluted to 1ug/mL and added metal ions to CB6-C diluents of different concentrations to make a final concentration of metal ions in each of a 10 mM. Then, a 50 uL metal ions diluent was added to the 96-well plate as test group, respectively. Additionally, we used PBS to dilute metal ions to a 10 mM and took a 50 uL diluted liquid and added to the 96-well plate as a negative control. Then, a 50 uL MRSA (105 CFU/mL) was added to each well and incubated at 37 °C for 18 h. The absorbance was measured by the OD at 600 nm and the minimum growth concentration was noted for evaluating bacterial growth. The experiment was performed three times.
Measurement of ROS release
The total reactive oxygen species (ROS) released from MRSA treated with the different concentrations of antibacterial protein CB6-C (8 µg/mL, 16 µg/mL, 32 µg/mL and 64 µg/mL) were probed with an ROS assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China), and performed according to the manufacturer’s instructions. No treatment with antibacterial protein CB6-C was performed as the negative control for ROS production. The fluorescence value of each sample was measured with an F4500 fluorescence spectrophotometer (emission λ = 525 nm, excitation λ = 488 nm) (Hitachi, Tokyo, Japan).
Measurement of adenosine triphosphate (ATP) and alkaline phosphatase (AKP) release
Intracellular ATP and AKP leak out when bacteria are destroyed. Therefore, we examined the amount of extracellular AKP and intracellular ATP to evaluate the effect of antibacterial protein CB6-C on MRSA [14, 15]. In brief, after the MRSA strain was cultured to logarithmic growth phase (OD at 600 nm = 0.5), it was centrifuged and washed twice with PBS (pH 7.4). The washed MRSA cells were treated with 1 × MIC of antibacterial protein CB6-C and incubated at 37 °C for 6 h, and the amount of extracellular AKP release was examined every hour. Similarly, MRSA cells were treated with 8 µg/mL to 64 µg/mL of antibacterial protein CB6-C for 1 h to examine the amount of intracellular ATP. The amount of extracellular AKP and intracellular ATP was measured using the ATP and AKP test kits (Jiancheng Biology Engineering Institute, Nanjingjiancheng, China) according to the manufacturer’s instructions.
Membrane permeability assay
Changes in membrane permeabilization were determined by measuring intracellular β-galactosidase activity as previously described. In short, MRSA in the mid-log phase was washed with PBS three times, diluted to 105 CFU/mL and an equal amount was added to a 96-well plate. Concurrently, different concentrations of antibacterial protein CB6-C (1 × MIC to 4 × MIC) were added to each well and incubated at 37 °C. In addition, the antimicrobial peptide LR18 (it has been reported to destroy MRSA cell membranes) stored in our laboratory was adjusted to a final concentration of 1 × MIC as a positive control [9]. The absorbance was measured by the OD at 420 nm and recorded for 1 h after every 10 min.
Preparation of protoplasts and MIC assay
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The preparation of protoplasts was carried out according to the method of Fan et al. [16]. In short, MRSA cells were cultured on MH broth medium to the logarithmic growth phase (OD at 600 nm = 0.5). A 5 mL of MRSA bacterial suspension was mixed with 5 mL of lysozyme (100 μg/mL) and incubated at 37 °C for 1 h. Then, the bacterial suspension was centrifuged at 4000 × g for 10 min at 4 °C, and the supernatant was discarded. Two washes with hypertonic buffer (0.1 mol/L phosphate buffer pH 6.0 and 0.8 mol/L Mannitol) were used to remove the enzyme, and protoplasts were suspended in a 5 mL of hypertonic buffer. The protoplast detachment was immediately observed under the microscope. The preparation of protoplasts was considered successful when more than 95% of the cells were gram-stained red. In addition, to evaluate the effect of antibacterial protein CB6-C on the cell membrane, MIC tests were performed on the prepared protoplasts according to the method outlined above in “Antibacterial spectrum and minimum inhibitory concentration” section. (MIC test methods), and untreated MRSA was used as the positive control.
Scanning and transmission electron microscopy
To investigate the morphological changes of MRSA cells after treatment with antibacterial protein CB6-C, we performed Scanning electron microscopy (SEM) according to a method outlined in a previous study [17]. In brief, MRSA of logarithmic growth phase (OD at 600 nm = 0.5) was diluted to 105 CFU/mL, added to a final concentration of 16 μg/mL antibacterial protein CB6-C (1 × MIC), and then incubated in the 6-well cell plate (containing polylysine-treated glass slides) at 37 °C for 3 h, with untreated MRSA cells used as a control. After incubation, the bacterial suspension was removed, and the polylysine-treated glass slides were fixed with 2.5% glutaraldehyde at 4 °C for 12 h, dehydrated with ethanol dilutions, dried and sprayed. The bacterial specimens were imaged using a FlexSEM 1000 SEM (JEOL, Hitachi, Tokyo, Japan).
For clearer observation of intracellular changes in MRSA, transmission electron microscopy (TEM) was performed according to the protocol described by Qin et al. [18] Briefly, cells were fixed with 2.5% glutaraldehyde as described above. After that, cells were osmicated in 2% osmium tetroxide for 4 h, dehydrated with ethanol solutions and embedded in epoxy resin. Finally, the sections were coated and stained using 2% uranyl acetate and lead citrate and observed with an E-1010 TEM (JEOL, Hitachi, Tokyo, Japan).
Competitive inhibition assay
To detect the effect of CB6-C on the cell wall main components (peptidoglycan, membrane teichoic acid, and Staphylococcal Protein A) of Staphylococcus aureus (S. aureus), CB6-C was diluted to different concentrations (4 µg/mL to 512 µg/mL), and the MIC assay was performed as described above in “Antibacterial spectrum and minimum inhibitory concentration” section. (MIC test methods). During the test, a 10 µg of equal volume of peptidoglycan, membrane teichoic acid, and Staphylococcal Protein A from S. aureus was added to each well of the 96-well plate to detect the effect of different proteins on the antibacterial activity of CB6-C.
Mouse infection models
To determine the treatment effect of antibacterial protein CB6-C in mice, we adopted the method based on previous studies by Song et al. [19] Briefly, a total of 30 BALB/c female mice were randomly transferred to three groups of cages (n = 10 per group), and each mouse was infected with a dose of 1.15 × 109 CFU MRSA in suspension via intraperitoneal injection (previous experimental results showed that the mortality rate of mice infected with this dose was higher than 80% within 48 h). The mice were treated with a specified intraperitoneal administration of PBS, antibacterial protein CB6-C (10 mg kg − 1), or antibacterial protein CB6-C (20 mg kg − 1) after one hour infection. The mice were observed for 48 h, and dead mice were removed to confirm the treatment effect of antibacterial protein CB6-C.
The mouse organ bacterial load test was the same as the treatment trial method, 40 mice were randomly divided into two groups (n = 20 per group) and infected intraperitoneally with 1.15 × 109 CFU MRSA in suspension. At 1 h postinfection, mice were treated with PBS and antibacterial protein CB6-C (20 mg kg − 1). After 48 h, the mice were euthanized, the heart, liver, spleen, lungs and kidneys were removed, and one part was washed with sterile PBS, soaked in 4% paraformaldehyde for fixation, and waited for subsequent hematoxylin-eosin (HE) staining. Other part of the organs tissue was washed with sterile PBS and homogenized in sterile PBS, and the bacterial loads in the different organs were counted.
Statistical analysis
One-way analysis of variance (ANOVA) was performed using SPSS v.22.0 software, followed by Tukey test. All data results were expressed as mean ± standard deviation. The mean values were considered significant different when *p < 0.05, **P < 0.01.
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