Materials. Egg yolk Lphosphatidylethanolamine (EYPE), egg yolk L-phosphatidyl-DLglycerol (EYPG), LPS purified from Escherichia coli O111:B4, were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3,3′-Dipropylthiadicarbocyanine iodide (diSC3-5), SYTO 9 and propidium iodide (PI) were supplied from Molecular Probes (Eugene, OR, USA). HyClone Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from SeouLin Bioscience (Seoul, Korea). The enzyme-linked immunosorbent assay (ELISA) kits for TNF‐α were procured from R&D Systems (Minneapolis, MN).
Bacterial strains. Bacterial strains were chosen to detect the minimal inhibitory concentration (MIC) of the peptides, as previously described45. Three strains of gram-positive bacteria (Bacillus subtilis [KCTC 3068], Staphylococcus epidermidis [KCTC 1917], and Staphylococcus aureus [KCTC 1621]) and three strains of gram-negative bacteria (Escherichia coli [KCTC 1682], Pseudomonas aeruginosa [KCTC 1637], and Salmonella typhimurium [KCTC 1926]) were procured from the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Methicillin-resistant Staphylococcus aureus (MRSA) [CCARM 3089, CCARM 3090, and CCARM 3095] and multidrug-resistant Pseudomonas aeruginosa strains (MDRPA) [CCARM 2095, and CCARM 2109] were obtained from the Culture Collection of Antibiotic-Resistant Microbes (CCARM) of Seoul Women's University in Korea. Vancomycin-resistant Enterococcus faecium (VREF) [ATCC 51559] was supplied from the American Type Culture Collection (Manassas, VA, USA).
Peptide synthesis and characterization. Peptides are synthesized using solid-phase peptide synthesis employing a fluorenylmethoxycarbonyl (Fmoc) protecting group strategy46. The peptides were purified by reversed-phase preparative HPLC on a C18 column (250 mm ⋅ 20 mm; Vydac) using an appropriate 0−90% H2O/CH3CN gradient in the presence of 0.05% trifluoroacetic acid. The purity (≥95%) and hydrophobicity were analyzed by reversed-phase analytical HPLC on C18 column (4.6 mm ⋅250 mm; Vydac). The molecular masses of purified peptides were determined by ESI-MS (electrospray ionization-mass spectrometry) (Framingham, MA, USA).
Bioinformatic analysis. The α-helical wheel plot, net charge and hydrophobic moments were calculated online using the HeliQuest server. The three-dimensional structure of Hybrid-LK was predicted online using I-TASSER server20.
Circular dichroism (CD) spectroscopy. CD studies were done in a JACSO-(J-715) spectropolarimeter (Jasco, Japan) with 0.1 mm quartz cuvette at 25ºC. The peptides were dissolved in 10 mM sodium phosphate buffer (pH 7.4), 50% TFE, and 30 mM SDS micelles. The percentage of α-helix of the peptides was calculated using the following equation, α-helix (%) = −100 × (θ222 + 3000)/33000.
Minimum inhibitory concentration (MIC). The minimal inhibitory concentrations (MICs) of the peptides against bacterial strains were determined via the broth microbroth dilution protocol recommended by the Clinical and Laboratory Standard Institute (CLSI)21,25. In brief, mid-logarithmic phase of bacteria was diluted with Mueller-Hinton broth (MHB) (Difco, USA) and added to a microtiter 96-well plate (2×106 CFU/well). A two-fold serial dilution of samples (concentration range: 1–64µM) was subsequently added, and the plate was incubated for 24 h at 37°C. The MIC (mg/L) was taken as the lowest concentration of the antimicrobial that inhibited the visible growth of the bacteria. All experiments were performed in triplicate and included growth and sterility controls.
Hemolytic activity. The hemolytic activity of the peptides was determined as the amount of hemoglobin released by the lysis of sheep red blood cells (sRBCs), as previously described45. Briefly, fresh sheep red bloods were washed with PBS and 4% blood solution was prepared in PBS. In a 96-well plate, 100 µl of varying concentrations of peptides were prepared. Another 100 µl of 4% blood solution was added to each well. The plate as then incubated for 1 h at 37°C. The plate was centrifuged and the OD450 of the supernatant was measured. 0.1% triton-X100 was taken a positive control and PBS was taken as a negative control.
Cytoplasmic membrane depolarization assay. The cytoplasmic membrane depolarization activity of the peptides was determined with the membrane potential-sensitive fluorescent dye, diSC3-5, as previously described33. Briefly, logarithmic growing S. aureus (KCTC 1621) cells were harvested and diluted to OD600 = 0.05 in 5 mM HEPES buffer (pH 7.4, containing 20 mM glucose). The cell suspension was further incubated with 0.4 µM diSC3-5 and 100 mM K+ until no further reduction of fluorescence. The fluorescence was recorded (excitation λ = 622 nm, emission λ = 670 nm) with a Shimadzu RF-5300PC fluorescence spectrophotometer (Kyoto, Japan). Subsequently, 3 ml of cell suspension was added to a 1 cm quartz cuvette and mixed with the peptides at their 2×MIC. Changes in the fluorescence were recorded from 0 to 500 s.
Dye leakage assay. Prepared calcein-entrapped large unilamellar vesicles (LUVs) were optimized using a previous method47. The negatively charged lipids composed of EYPE/EYPG (7:3, w/w) were dissolved in chloroform, dried with a stream of nitrogen and resuspended in dye buffer solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, and 0.1 mM EDTA, pH 7.4). The suspension was subjected to 10 freeze-thaw cycles in liquid nitrogen and extruded 21 times through a LiposoFast-Extruder (Avestin, Inc., Canada) equipped with filters of 100 nm pore size. Untrapped calcein was removed from the liposome by gel filtration on a Sephadex G-50 column Calcein leakage from liposomes was monitored at room temperature by measuring fluorescence intensity at an excitation wavelength of 490 nm and emission wavelength of 520 nm on a Shimadzu RF-5300PC fluorescence spectrophotometer (Kyoto, Japan). Complete dye release was obtained using 0.1% Triton X-100.
Cytotoxicity against RAW264.7 macrophage cells. The cytotoxicity of the peptides against mouse macrophage RAW264.7 cells was assessed by MTT assay48. Briefly, RAW264.7 cells were cultured in DMEM (Gibco) with 10% FBS in a humidified atmosphere containing 5% CO2 at 37°C. The cells were added to 96-well plates at a final concentration for 2×104 cells per well in DMEM and cultured overnight. TZP4 was then added and incubated for 48 hr. MTT (50 µL, 0.5 mg/mL) was added to the 96-wellplate and incubated at 37°C for 4 hr. Subsequently, 150 µL of DMSO was added to dissolve the formed formazan crystals after the supernatant was discarded, and the OD was measured using a microplate reader (Bio-Tek Instruments EL800, USA) at 550 nm. Cell viability was expressed as (A550nm of treated sample) / (A550nm of control) 100%.
Measurement of nitric oxide or tumor necrosis factor-α release from LPS-stimulated RAW264.7 cells. Peptide-induced inhibition of nitric oxide (NO) and proinflammatory cytokine, tumor necrosis factor (TNF)‐α production in LPS-stimulated macrophage cells were measured as previously described49. In brief, RAW264.7 murine macrophage cells (2 ⋅106 cell/mL) were plated and adhered to a 96 well plates (100 mL/well) and stimulated with LPS from E. coli O111:B4 (20 ng/mL) in the presence or absence of peptide for 24 h. After 24 h incubation, the culture supernatant was collected for enzyme linked immunosorbent assay (ELISA) to detect the level of nitric oxide (NO) and inflammatory cytokine TNF-α. The nitrite level was determined using Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid). Release of TNF‐α was detected using DuoSet ELISA mouse TNF-α (R&D Systems, Minneapolis, USA) according to the manufacturer’s protocol.
Reverse-transcription polymerase chain reaction (RT-PCR). RT-PCR was performed as previously described45. RAW264.7 cells were plated into 6-well plates at a concentration of 5 × 105 cells/well and stimulated with E. coli O111:B4 LPS (20 ng/mL) in the presence or absence of the peptides. After incubation of 3 h (for TNF-α) and 6 h (for inducible nitric oxide synthase (iNOS), total RNA was extracted using TRIzol® reagent (Invitrogen) and RNA concentration quantified using Nanodrop spectrophotometer (BioDrop, UK). cDNA was synthesized from 2 µg of total RNA using Oligo-d(T)15 primers and PrimeScript Reverse Transcriptase kit (Takara, Japan) according to the manufacturer’s protocol. The cDNA products were amplified using following primers: iNOS (forward 5´-CTGCAGCACTTGGATCAGGAACCTG-3´, reverse 5´-GGGAGTAGCCTGTGTGCACCTGGAA-3´); TNF-α (forward 5´- CCTGTAGCCCACGTCGTAGC-3´, reverse 5´-TTGACCTCAGCGCTGAGTTG-3´) and GAPDH (forward 5´-GAGTCAACGGATTTGGTCGT-3´, reverse 5´- GACAAGCTTCCCGTTCTCAG-3´) (Jantaruk, Roytrakul, Sitthisak, & Kunthalert, 2017). The PCR amplification was carried out at initial denaturation at 94°C for 5 min, followed by forty cycles of denaturation at 94°C for 1 min, annealing at 55°C for 120 sec and extension at 72°C for 1 min, with a final extension at 72°C for 5min. The PCR products were separated by electrophoresis and visualized under UV illumination.
LPS-binding assay. The LPS‐binding ability of the peptides was determined by a BODIPY‐TRcadaverine (BC) displacement assay50,51. Briefly, LPS from E. coli O111:B4 (25 µg/ml) was incubated with BC (2.5 µg/ml) and peptide (1‐32 µM) in Tris buffer (50 mM, pH 7.4) for 4 hr. A volume of 2ml of this mixture was added to a quartz cuvette. Fluorescence was recorded at an excitation wavelength of 580 nm and an emission wavelength of 620 nm with a Shimadzu RF‐5301 PC fluorescence spectrophotometer (Shimadzu Scientific Instruments). The percentage fluorescence was calculated using formula: %ΔF (AU) = [(Fobs − F0)/(F100 − F0)] × 100, where Fobs is the observed fluorescence at a given peptide concentration, F0 is the initial fluorescence of BC with LPS in the absence of peptides, and F100 is the BC fluorescence with LPS cells upon the addition of 10 µg/ml polymyxin B.
Biofilm eradication assay (MBEC). Biofilm eradication, determined as the MBEC of the peptides, was assessed according to methods from the literature using the Calgary Biofilm Device (Innovotech, Edmonton, Canada)52,53. Briefly, 1 × 106 CFU/mL of bacteria were suspended in 150 µL of appropriate nutrient media (LB media) and placed in 96-well microtiter plates with peg lids (Innovotech, Edmonton, Canada; product code: 19111) to establish biofilms. Plates were sealed with parafilm and incubated at 37°C for 24 h in a shaking incubator at 110 rpm. Lids of the plates were then removed, rinsed with 0.01 M PBS, and transferred to sterile 96-well plates containing serial dilutions of the peptides: the final volume with the media was 200 µL/well. Plates were then incubated at 37°C for 24 h in a shaking incubator at 110 rpm. After 24 h of treatment, the peg lid of each plate was removed, rinsed with buffer, and transferred to a recovery plate containing 200 µL of nutrient media. Recovery plates were thereafter sonicated in a water bath for 10–15 min to dislodge biofilms. Peg lids were removed and plates were incubated overnight (for 24 h) at 37°C in a shaking incubator at 110 rpm to recover viable bacteria, resulting in turbidity. The MBEC values were recorded as the lowest concentration resulting in eradication of the biofilm (i.e., no turbidity after the final incubation period relative to sterility controls). Experiments were performed in triplicates, and the median value of each experiment was presented.
Confocal laser scanning fluorescence microscopy (CLSM). MDRPA (1×106 CFU/mL) was cultured in 24-well plates containing discs placed in MHB-glucose medium, for 24 h to form biofilms. Discs with planktonic cells were washed with 1× PBS thrice and placed in fresh 24-well plates containing Lf-KR (MBEC: 16 µΜ), and plates were incubated for 6 h. Discs were removed, washed twice with 1 × PBS, and concomitantly stained with 6.7 µM SYTO 9 and 40 µM PI. After incubation in the dark at 37°C for 30 min, planar images of biofilm mass in the discs were visualized using CLSM (Zeiss LSM 710 Meta, ZEISS Microscopy, Jena, Germany), and analyzed using ZEN 2009 Light Edition software (ZEISS Microscopy, Jena, Germany).