Bacterial strain, antibiotic, and mice
S. aureus ATCC25923 was kindly provided by Bin Tang at Jilin University. L. monocytogenes CICC21634 was obtained from China Center of Industrial Culture Collection (CICC, China). S. typhimurium SL1344 and P. aeruginosa GIM1.551 were isolated from clinical cases and maintained in our laboratory. NIH 3 T3 cells were kindly provided by Dr. Lu at Nanjing Medical University. Thiazolyl blue tetrazolium bromide (MTT), Dulbecco’s modified eagle medium (DMEM), and dimethyl sulfoxide (DMSO) were purchased from Sijiqing Biotech (China). Ceftiofur sodium, ampicillin sodium, benzylpenicillin potassium, sodium pentobarbital, lysogenic broth (LB) medium, brain heart infusion (BHI) medium, tryptic soy broth (TSB) medium, Mueller–Hinton broth (MHB) medium, fetal bovine serum, Triton X-100, and propidium iodide (PI) were purchased from Procell Corporation (Wuhan, China). All other chemical reagents used were analytical grade. Three hundred specific pathogen-free (SPF) BALB/c mice (aged 4–6 weeks, 20 ± 2 g; 50% male and 50% female) were purchased from Henan Province Experimental Animal Center (Henan, China). All procedures performed in studies involving animals complied with the Animal Ethics Procedures and Guidelines of the People’s Republic of China.
Peptide synthesis
PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) were synthesized by Fmoc solid-phase peptide synthesis at Shanghai Apeptide Biological Technology Co., Ltd. (Shanghai, China). All peptides were purified by HPLC to high purity (≥95%), and the synthesized peptides were identified by MS. The HPLC and MS protocol are as follows. HPLC experiments of PMAP-37 and Chol-37(F34-R) were carried out on Waters 2690 and HPLC experiment of PMAP-37(F34-R) was carried out on SHIMADZU LC-2010HT. The instruments were equipped with a Waters SinoChrom ODS-BP column (5 μm, 4.6 × 250 mm) at a flow rate of 1.0 mL/min using a gradient of 5–65% acetonitrile in water with 0.1% TFA as the mobile phase. UV detection was performed at a wavelength of 220 nm. MS experiments of PMAP-37 and Chol-37(F34-R) were carried out on SHIMADZU LCMS-2010EV and MS experiment of PMAP-37(F34-R) was carried out on Agilent LCMS-6125B. The ESI source conditions were as follow. Nebulizer gas flow: 1.5 L/min; CDL temperature: 250 °C; capillary voltage: 1500 V; T. flow: 0.2 mL/min. The mass scan was in the range of m/z 400–2000.
Inhibition zone assays
The antibacterial activity of Chol-37(F34-R) was detected using the Kirby-Bauer diffusion method [40]. Four bacterial strains were used as experimental strains: L. monocytogenes CICC21634 cultured with BHI, P. aeruginosa GIM1.551 cultured with TSB, S. aureus ATCC25923, and S. typhimurium SL1344 cultured with LB medium. Briefly, the bacterial cells were cultured for 12 h at 37 °C in appropriate culture media, and the bacterial cell suspension was spread on the corresponding solid medium. Then, the disks containing 10 μg peptide or ceftiofur sodium were attached to the solid medium and incubated at 37 °C for 18 h. Lastly, the diameter of the inhibition zone was measured.
Minimal inhibition concentration (MIC) assay
The MIC values of Chol-37(F34-R) were determined using the microdilution method of the Clinical and Laboratory Standards Institute [41]. Antibacterial intensity was tested in MHB against S. aureus ATCC25923, L. monocytogenes CICC21634, S. typhimurium SL1344, and P. aeruginosa GIM1.551. The tested strains were cultured for 12 h at 37 °C. Then, the bacterial inoculum concentration was adjusted to 1 × 106 CFU/mL. Twofold serial dilutions covering different concentrations from 0.0039 to 256 μg/mL for each peptide were added to media containing cultures of the tested strains in 96-well plates, which were incubated at 37 °C for 18 h. The MIC values were determined by evaluating the OD600nm of the cultures.
Biofilm inhibition assay
Biofilm inhibition assay was performed as described previously to investigate the biofilm inhibition capability of Chol-37(F34-R) [42]. The assay was conducted in TSB medium supplemented with 0.5% glucose against S. aureus ATCC25923, TSB medium against S. typhimurium SL1344, and BHI medium against P. aeruginosa GIM1.551. Briefly, 200 μL 1 × 106 CFU/mL bacterial cells was cultured at 37 °C for 24 h, in 96-well plates, with or without PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) (0.25–128 μg/mL). Planktonic bacteria were removed, and the wells containing biofilm were washed thrice with sterile PBS solution. Subsequently, 200 μL methanol (99%) was added per well, and the wells were fixed for 20 min. After aspiration, the plates were allowed to dry. The biofilm was stained with crystal violet (0.1%) for 10 min, and the excess colorant was gently eliminated by three successive washings with sterile PBS. The stain was resolubilized in 95% ethanol, and the absorbance was measured at 620 nm. Biofilm formation inhibition rate = [1 − OD620nm (peptides)/OD620nm (control)] × 100%.
Biofilm eradication assay
Biofilms of S. aureus ATCC25923, S. typhimurium SL1344, and P. aeruginosa GIM1.551 were grown in 96-well plates by adding 200 μL bacteria (1 × 106 CFU/mL) in the medium. After incubation at 37 °C for 24 h, the wells were washed thrice with PBS. The 128 μg/mL PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) samples were prepared in fresh 96-well plates using the same media. Then, 200 μL of the suspension was transferred to the plate containing biofilm. A total of 200 μL sterile medium was added to the negative control wells. The plates were incubated at 37 °C for 1 h. After incubation, the biofilm mass was determined by measuring the absorbance after applying crystal violet stain as described above. Biofilm mass % = OD620nm (peptides)/OD620nm (control) × 100%. The biofilms were solubilized with 200 μL 0.1% Triton X-100 to determine the number of viable bacteria in the biofilm. Following a 10-fold serial dilution in PBS, the bacterial cells were spread on agar plates and incubated at 37 °C for 18 h and subsequently counted.
Permeability assay
The membrane permeabilizing ability of Chol-37(F34-R) was detected using the PI uptake assay. PI was used as a fluorescent indicator to investigate the permeability of the bacterial membrane effects of Chol-37(F34-R). PI is a fluorescent dye that could enter dead bacteria and combine with DNA to emit red fluorescence, at excitation and emission wavelengths of 535 and 615 nm, respectively. The mid-logarithmic culture of S. aureus ATCC25923, L. monocytogenes CICC21634, S. typhimurium SL1344, and P. aeruginosa GIM1.551 was centrifuged at 2000 g for 5 min and resuspended in PBS (1 × 108 CFU/mL). The bacteria were treated with final concentrations of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) (0.0313 μg/mL for S. aureus ATCC25923, 4 μg/mL for L. monocytogenes CICC21634, 4 μg/mL for S. typhimurium SL1344, 2 μg/mL for P. aeruginosa GIM1.551) at 37 °C for 20 min. PBS was added to the control assay. Subsequently, an equal volume of PI (200 μg/mL) was added and the samples were stained in the dark for 10 min. The suspensions were added to the slides with cover slips for immobilization. The bacterial cells were imaged by fluorescence inverted microscope (Axio Observer A1, Zeiss). The microscopic images were captured by ZEN (2.6 version).
pH stability assay
To investigate the effect of pH on the antibacterial activity of Chol-37(F34-R), three peptides (1 mg/mL) with different pH buffers (pH 2–13) were incubated at 37 °C for 4 h. Then, the treated peptides were tested separately in inhibition zone assays against S. aureus ATCC25923 or P. aeruginosa GIM1.551. Each standard disk contained 10 μg peptide. The inhibition zone diameters of peptides were measured.
Salt ion and serum stability assay
The effects of salt ion and serum on the antibacterial activities of Chol-37(F34-R) were investigated by MIC assay. S. aureus ATCC25923 and P. aeruginosa GIM1.551 were used as the tested strains. The MIC values were determined at recommended doses of NaCl (8.766 g/L), CaCl2 (0.039 g/L), and fetal bovine serum (20%) [43].
Thermal stability assay
To test the thermal stability of Chol-37(F34-R), the three peptides (1 mg/mL) were boiled for 0, 10, 20, 30, 40, 50, 60, 90, and 120 min. Then, the treated peptides were tested separately in inhibition zone assays against S. aureus ATCC25923 or P. aeruginosa GIM1.551. Each standard disk contained 10 μg peptide. The inhibition zone diameters of peptides were measured.
Hemolytic assay
The hemolytic activity of Chol-37(F34-R) was investigated as described previously [44]. Mouse erythrocytes were diluted to a final suspension concentration of 8% (v/v). Then, 100 μL erythrocyte suspension was incubated with 100 μL peptides at different concentrations ranging from 2.5 to 1280 μg/mL. PBS and 0.2% Triton X-100 were used as negative and positive controls, respectively. After 1 h of incubation, the tested samples were centrifuged for 5 min at 1000 g. Lastly, the absorbance of the supernatant of tested samples was measured at 570 nm. The percentage of hemolytic was obtained using the following formula: Hemolysis rate = [OD570nm (peptides) − OD570nm (PBS)]/[OD570nm (Triton X − 100) − OD570nm (PBS)] × 100 % .
Cytotoxicity assay
The cytotoxicity of Chol-37(F34-R) was assessed by MTT assays as described previously [45]. NIH 3 T3 cells were seeded in a 96-well with a density of 1× 104 cells per well and incubated in 37 °C for 24 h. Afterward, the cell medium was replaced by fresh DMEM containing various concentrations of peptides (2.5–1280 μg/mL). After 24 h incubation, 20 μL MTT agent (5 mg/mL) was added to each well and incubated for 4 h. Then, the supernatant of each well was replaced with 150 μL DMSO. The absorbance was measured by a microplate reader (FlexStation III) at 570 nm.
Therapeutic analysis of P. aeruginosa GIM1.551-infected mouse knife injury model
The P. aeruginosa strain GIM1.551 was used to build knife wound model of mouse as described elsewhere with minor modifications [46]. A total of 30 mice were randomly divided into six groups. Briefly, the fur on the back of each mouse was removed by shaving. A 1.5 cm linear knife wound was made on the back of each mouse and infected by direct seeding with bacterial suspensions (1 × 108 CFU/mL, 50 μL). One day postinfection, the wounds were administered with PMAP-37 (10 μL, 1 μg/μL), PMAP-37(F34-R) (10 μL, 1 μg/μL), Chol-37(F34-R) (10 μL, 1 μg/μL), ampicillin sodium (10 μL, 1 μg/μL), or PBS (10 μL), and the treatment program was performed once per day for 7 days. The mice with no infection and treatment were used as blank control. The wounds of mice were wrapped in gauze bandages that were changed every day. Seven days after treatment, mice were euthanized with 120 mg/kg intraperitoneal (i.p.) sodium pentobarbital and their wound tissues were collected. The wound tissues were homogenized with a basic homogenizer. Following a 10-fold serial dilution in PBS, the homogenates were spread on TSB agar plates. The bacterial burden was determined after incubation at 37 °C for 18 h.
Therapeutic analysis of S. aureus ATCC25923 or P. aeruginosa GIM1.551-infected mouse abscess model
The abscess model of mouse was built against S. aureus ATCC25923 or P. aeruginosa GIM1.551 as previously described with minor modifications [47]. Twelve groups of 60 mice (5 mice per group) were randomly divided into PMAP-37, PMAP-37(F34-R), Chol-37(F34-R), ampicillin sodium (against S. aureus ATCC25923-infected mouse abscess model), benzylpenicillin potassium (against P. aeruginosa GIM1.551-infected mouse abscess model), PBS, and blank groups. The fur on the back of each mouse was shaved. Bacterial suspensions (1 × 108 CFU/mL, 50 μL) were injected into the right side of the dorsum underneath the thin skeletal muscle. After 1 h of inoculation, the infected areas were directly injected with PMAP-37, (10 μL, 1 μg/μL), PMAP-37(F34-R) (10 μL, 1 μg/μL), Chol-37(F34-R) (10 μL, 1 μg/μL), antibiotic (ampicillin sodium 10 μL, 1 μg/μL or benzylpenicillin potassium 10 μL, 1 μg/μL), or PBS (10 μL). The treatment program was conducted once per day for 3 days. Mice in the blank group were not challenged and treated. Three days after treatment, mice were euthanized with 120 mg/kg i.p. sodium pentobarbital, and their skin abscesses were harvested and homogenized as described above to assess the bacterial burden.
Therapeutic analysis of S. aureus ATCC25923-infected mouse peritonitis model
The dissemination of S. aureus ATCC25923 to target organs was assessed in the peritonitis model. The assessment was performed on the basis of the method described previously with minor modifications [48]. Six groups of 60 mice (10 mice per group) were randomly divided into PMAP-37, PMAP-37(F34-R), Chol-37(F34-R), ampicillin sodium, PBS, and blanks group. Bacterial suspension (1 × 108 CFU/mL, 100 μL) was intraperitoneally injected into mice. After 2 h inoculation, PMAP-37 (20 μL, 1 μg/μL), PMAP-37(F34-R) (20 μL, 1 μg/μL), Chol-37(F34-R) (20 μL, 1 μg/μL), ampicillin sodium (20 μL, 1 μg/μL), or PBS (20 μL) was intraperitoneally injected into the mice, and the treatment program was implemented once per day for 3 days. Mice in the blank group were not challenged and treated. After 3 days of treatment, mice were euthanized with 120 mg/kg i.p. sodium pentobarbital, and the livers and spleens were collected, weighed, and placed in 1 mL PBS. Aliquots of diluted homogenized tissues were spread on LB agar, on which the bacterial burden was determined for analysis. Furthermore, liver and spleen sections were prepared to observe pathological damage as previously reported [49]. The liver and spleen sections were scanned by microscope slide scanner (Pannoramic DESK, 3D HISTECH) and viewed by Caseviewer (C.V 2.3 version). Pathological changes were scored as follows: a score of 1 indicated no pathology, 2 indicated perivascular infiltrates, 3 indicated perivascular and interstitial infiltrates affecting < 20% of the section, 4 indicated perivascular and interstitial infiltrates affecting 20 to 50% of the section, and 5 indicated perivascular and interstitial infiltrates affecting > 50% of the section [50, 51].
Statistical analysis
The data are presented as means ± standard deviation (SD) of the mean. Statistical analyses were performed using one-way ANOVA F-statistics, and differences were considered significant at P < 0.05 or P < 0.01.