1.1 Bacterial strains and growth conditions
Staphylococcus aureus AB94004, Staphylococcus aureus ATCC25923, Staphylococcus aureus ATCC6538, Escherichia coli AB94012, Escherichia coli DH5a, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC9027, Pseudomonas aeruginosa PAO1 were used in this study and purchased from the China Center of Type Culture Collecting (CCTCC). Bacterial were cultured to the exponential phase in Luria-Bertani (LB) broth medium and incubated at 37℃ with continuous shaking at 250rpm. Three replicates at least were set for each group, and each experiment was repeated at least twice in the following assays except animal experiment.
1.2 Peptide synthesis
The peptides used in this study (Table 1) were synthesized by GL Biochem (Shanghai, China) with amidated C-terminus with a purity of >95%.
1.3 Animals and ethics statement
All animal procedures were performed and approved by the Animal Care Committee of the Henan University of Science and Technology, which meets the NIH guidelines for the care and use of laboratory animals. The BALB/c male mice were used in the experiment with a body weight of 20-30g. They were housed and bred under standard conditions (25±2°C, 12-h light/dark cycle) with free access for food and water.
1.4 Antimicrobial assays
The MICs of the peptides against the tested microorganisms were determined by using a protocol described by the Clinical and Laboratory Standards Institute (CLSI) Briefly, bacteria were cultured at 37℃ with continuous shaking at 250rpm overnight to the exponential phase in Luria-Bertani (LB) broth medium, then diluted to 105 -106 CFU/ml (CFU: colony-forming unit) in LB medium for use. 100ml of the above prepared bacterial suspensions was added into the 96-well plate, and then 100ml of equal dilution peptide solution was added into each wall. The final concentrations peptides in each well were 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625mg/mL, respectively. PBS was added in the control well. The samples were incubated at 37℃ for 24h. The lowest concentration of peptide that completely inhibited bacterial growth was deemed to be the MIC value.
1.5 Thermal stability assays
Thermal stability of the peptides was measured by the antimicrobial assays mentioned above after the peptides were placed at 60℃ for 24h.
1.6 Checkerboard assay
Checkerboard assay [17] was used to determine the synergistic effect of antimicrobial peptides and conventional antibiotics including ciprofloxacin (Solarbio life science, C9371), doxycycline (Solarbio life science, SD8430), ceftriaxone (Solarbio life science, C7780), and neomyein (Solarbio life science, N8090) on Escherichia coliAB94004 and P. aeruginosa PAO1. FICI is then calculated according to the following formula: FIC index (FICI) = FIC A + FIC B = A / MIC A + B / MIC B. MIC A and MIC B represent the minimum inhibitory concentration (MIC) values of peptide A and antibiotic B alone. A and B represent the optimal concentrations of peptide A in combination with antibiotic B. FICI ≤ 0.5 indicated synergistic antibacterial effect; 0.5 < FICI ≤ 4 indicated no correlation between the two; FICI ≥ 4 indicates antagonism [18].
1.7 Hemolytic activity
Blood was got from the mouse’s heart after anesthesia. After centrifugation at 3000rmp for 10mins, the supernatant was discarded to obtain fresh red blood cells (RBC). The RBC were washed with normal saline 3 times and then re-suspended with a concentration of 2% (V/V). 100ml of RBC suspension and different concentrations of antimicrobial peptides both added to the wall of 96-well plate and incubated at 37℃ with gentle shaking for 1h. After centrifugation for 10min at 1000rpm, the supernatant was transferred to a new 96-well plate and the absorbance was measured at 490nm. Normal saline and 1% Triton X-100 were set as the negative and positive controls, respectively. Hemolysis% = (A sample – A negative) / (A positive − A negative) × 100%. A: absorbance at 490 nm.
1.8 Cytotoxicity assay
The 293T and HELA cell lines were used for cytotoxicity of the antimicrobial peptide. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 100 U/ml of penicillin, 100μg/ml of streptomycin double antibiotics and 10%FBS (Gibco, Grand Island, NY, USA) and cultured in a 37℃ incubator with 5%CO2. Cytotoxicity of the peptides against the cell lines was evaluated by the MTS assay. First, 7000 cells were seeded in each wall of the 96-wall plant and cultured overnight. Then 100ml of antimicrobial peptide with different concentrations were added for co-cultured for another 24h in incubator. Finally, 20µl of MTS (Promega, Madison, WI, USA) was added to each well and incubated for 1h to measure the absorbance at 590nm. PBS and 0.1% Triton X-100 were set as negative and positive controls, respectively. Viability% = (A sample − A positive)/ (A negative − A positive) × 100%. A: absorbance at 590 nm.
1.9 Time-killing kinetics
Bacteria were cultured overnight to exponential growth stage and diluted with fresh LB medium to a final concentration of 105-106 CFU/ml. Peptide solution were added in the experimental groups with final concentrations of 1MIC, 2MIC and 4MIC. PBS was added to the control group. The above bacterial liquid was placed in a 37℃ incubator. Same amount of medium was taken out in 0, 15, 30, 45, and 60mins respectively, then dilution and plated. The plates cultured at 37℃ overnight and colony count the next day [19] [20].
1.10 Drug resistant assays
Bacteria at logarithmic growth stage were collected and suspended with LB at a final concentration of 109CFU/ml. Antimicrobial peptides and antibiotics including ciprofloxacin (Solarbio life science, C9371), doxycycline (Solarbio life science, SD8430) and ceftriaxone (Solarbio life science, C7780) were added into the above bacterial solution. Antimicrobial peptides and antibiotics mentioned above were added to the bacteria that growing at the concentration of 1/2MIC after the bacteria were suspended with LB at a final concentration of 109 CFU/mL again. Fold MIC = MIC n / MIC 1 (n indicates days). MIC tests were performed for 15 days consecutively.
1.11 Biofilm formation and viable bacterial load in biofilm
Bacteria at logarithmic growth stage were suspended with LB at a final concentration of 105-106 CFU/ml. The above prepared bacteria solution 100ml contained CT-K3K7 with final concentration of 0MIC, 1/2MIC, 1MIC, 2MIC, 4MIC was added to each well of 96-well plate and cultured in an incubator for 24 hours. The viable bacterial load in biofilm of some wells were diluted and plated, the number of bacterial colonies were observed after being cultured at 37℃ overnight [21]. The bacterial solution of the remaining wells was added with 10ml of XTT detection solution (Sigma Chemical Co., St. Louis, Mo.) and detected absorbance at 490nm to observe the formation of biofilms.
1.12 Subcutaneous abscess model
BALB/C male mice were randomly divided into subcutaneous abscess model group, positive control group and CT-K3K7 group. First, the skin on the back of mice was prepared and disinfected in advance. Then 50ml of S. aureus AB94004 (1010) or P. aeruginosa PAO1 (109) was injected subcutaneously to establish subcutaneous abscess model. One hour later, CT-K3K7 (0.5mg/ml, 50ml) was directly injected into the abscess site; same valume of normal saline was injected into the model group; Vancomycin (0.5mg/ml, 50ml) was used against S. aureus abscess model and ciprofloxacin (0.5mg/ml, 50ml) was used against P. aeruginosa abscess model as positive control. The drug was administered continuously for 3 days. The subcutaneous abscess area (Abscess area=length×width) was observed and measured after the mice were euthanasia on the fourth day. Bacterial colony number was observed with the coating plate after grinding half of the abscess. Another half of the abscess tissue was placed in 4% paraformaldehyde solution to observe the infiltration of inflammatory cells under microscope after embedding and sectioning.
1.13 PI staining
Bacteria in logarithmic phase were suspended with LB as a suspension with a final concentration of 109 CFU/ml. Antimicrobial peptides with a final concentration of 0MIC, 1/2MIC, 1MIC, 2MIC, 4MIC and propidium iodide (PI, Solarbio life science, Beijing, China) with a final concentration of 10 mg/mL were both added into the above bacterial solution. The control group was added with normal saline. The solutions were incubated at room temperature without light for 15-30 minutes, then discarded the supernatant after centrifuge at 3700rpm for 5mins. After washing with normal saline, bacteria with PI staining were observed under fluorescence microscope to evaluate the effect of peptide on bacterial membrane integrity.
1.14 DNA binding assay
After being dissolved in DNA binding buffer, CT-K3K7 was added to the DNA of plasmid PET28a, double-enzyme (EcoR I and Xho I) cut PET28a and salmon sperm. The final mass ratio of CT-K3T3 to DNA was 40:1, 20:1,10:1,5:1,0:1. After the solutions were incubated for 30mins, electrophoresis with 1% agarose gel was used to detect the binding phenomena of antimicrobial peptide and DNA in different forms. VU gel imaging was used to observe and take photos.
1.15 Statistical analysis
The data are expressed as the mean ± SEM. All statistical analyses were analyzed using the GraphPad Prism 6 software. Data were statistically analyzed by analysis of one-way variance (ANOVA) between groups. P < 0.05 was considered statistically significant.