Materials
DEAE-Sepharose Fast Flow and Sephadex G-25 columns were obtained from Pharmacia (Uppsala, Sweden). Bacterial media de Man-Rogosa-Sharpe (MRS) and Mueller-Hinton (MH) were purchased from Becton–Dickinson, Spark, USA. Agar was purchased from Daejun Chemicals and Metals Co, Gyeonggi-do, South Korea. All other reagents were of the extra pure grade. Strain CBSYD1 was isolated from fermented kimchi.
Culture media for YD1 production
The impact of various nutrient sources (carbon, nitrogen, and metal ion) on the antimicrobial compound production was determined and media optimization was performed according to our previous report (Cho et al. 2012). Fermentation was carried out in 50 mL media in 250-mL Erlenmeyer flasks with constant shaking at 160 rpm. Zone of inhibition was observed against Mycobacterium smegmatis ATCC 9341 at every step of media optimization. Commercially available MRS and MH broth media were used as control media.
Bacterial strain isolation and identification
Cabbages, from different provinces of Korea, were collected and processed for biochemical and molecular identification of microorganisms. The strain identification, based on morphological characteristics, was made according to Bergey’s manual of systematic bacteriology (Lechevalier 1989). Furthermore, the identification was confirmed by 16S rRNA sequence analysis and phylogenetic tree.
The nucleotide sequence of strain CBSYD1 was submitted to the GenBank (ncbi.nlm.nih.gov/Genbank) under the accession no. KY062987.
Antimicrobial activity
A filter paper disc (8 mm, Toyo Roshi Kaisha, Japan) saturated with antimicrobial sample (40 µL) was placed on the surface of petri dish (87 mm × 15 mm) containing Mueller Hinton Agar (MHA). The plate was incubated at 37 °C, and a clear zone of inhibition surrounding the paper disc was measured in millimeter (mm).
An arbitrary unit per milliliter (AU/mL) was defined as the reciprocal of the dilution after the last serial dilution that resulted in an inhibition zone. The titer of the antimicrobial substance solution, in AU/mL, was calculated as (1000/d) D, where D was the dilution factor, and d was the dose, the amount of antimicrobial substance solution added to each spot. AU and the zone of inhibition were measured against Mycobacterium smegmatis ATCC 9341 while optimizing CBSYD1 media.
The minimal inhibitory concentration (MIC) was determined according to the method described by Weigand et al. (2008).
Purification of YD1
CBSYD1 was cultured for 36 h in optimized media (1% peptone, 2% maltose, and 0.01% CaCl2). The cultured supernatant was mixed with ammonium sulfate (30–80% saturation) and kept at 4 °C with overnight stirring. The precipitate was collected by centrifugation at 10,000 rpm for 30 min and re-dissolved in 10 mM Tris–HCl buffer (pH 7). The dialyzed sample was applied to a DEAE-Sepharose Fast Flow column (2.5 × 14 cm) pre-equilibrated with 10 mM Tris–HCl buffer, pH 7. The column was washed with the same buffer and eluted with a linear gradient of KCl (0–1 M). Fractions of 3 mL were collected at a flow rate of 0.3 mL/min. Active fractions were pooled, concentrated, and further purified with Sephadex G-25 column (1.5 × 28 cm) using the same buffer system.
Electrophoresis and in-situ analysis
The molecular weight of peptide was determined by tricine SDS-PAGE (Schägger 2006). The in situ analysis was performed against indicator organism (~5 × 105 cfu/mL) by overlaying the processed gel from tricine SDS–PAGE [after washes with 50 mM Tris/HCl buffer (pH 7.5) containing 2.5% Triton X-100 for several times] on 0.6% agar on Mueller–Hinton (DIFCO, USA) media and incubated at 37 °C.
Amino acid sequencing and computational analysis
Amino acid sequence of YD1 was determined by Edman degradation using a Procise Model 492 protein sequencer (Applied Biosystems, CA, USA). The amino acid sequence was analyzed using BLAST search against GenBank (http://www.ncbi.nlm.nih.gov/BLAST) and Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php). The 3D structure projection was predicted by I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) under the job ID S281576.
Stability of YD1
The thermal stability of YD1 samples was determined by exposure to 20, 40, 60, 80 and 100 °C for 30 min and to 121 °C/105 kPa for 15 min before analyzing the residual activity. Similarly, pH stability was determined over a range of pH 2–10 using 1 M NaOH or HCl. The effect of protease enzyme on YD1 stability was determined at two different enzyme concentrations (1 and 2 mg/mL).
Cytotoxicity
Cytotoxicity assay was performed according to our previous report by Choi et al. (2016). The murine macrophage cell lines Raw 264.7 were seeded in 96-well plates and 24 h later, treated with purified YD1, concentration ranging 8–120 µg/mL.
Synergism or antagonism of YD1 with antibiotics
YD1 was investigated for the interaction with antibiotics such as erythromycin (a protein synthesis translocation inhibitor), ceftriaxone sodium (a cell wall synthesis inhibitor), and a quinolone (ciprofloxacin) that interferes with DNA gyrase supercoiling.
Log phase-grown E. coli and MRSA were cultured in MHB at 37 °C and diluted to a final inoculation density of 1 × 105–1 × 106 cfu/mL in a total of 200 µL. The inhibition pattern indicates the interaction between the two compounds and the method enables the calculation of a fractional inhibitory concentration index (FICI), a numerical interpretation of the type of interaction displayed.
For wells containing the lowest inhibitory combination of drugs, a fractional inhibitory concentration (FIC) is derived for each well from the following calculation:
$$\begin{aligned} &= \frac{\text{MIC of compound A with B}}{\text{The MIC of compound A alone}}+ \frac{\text{MIC of compound B with A}}{\text{The MIC of compound B alone}}\end{aligned}$$
The FIC Index of ≤0.5 was considered to indicate synergism, a value ≥4 to indicate antagonism and all values >0.05 to <4.0 indicated an indifferent interaction (Ji 1996; Pasquale and Tan 2005; Williams 2001).
Time-kill interaction between an antibiotic and YD1
Time-kill assays were conducted with concentrations corresponding to the MIC values of YD1 and erythromycin for reference strain, E. coli. Concentrations ranging from 8 to 256 µg/mL of YD1 or erythromycin were added to a bacterial suspension (1 × 105–1 × 106 cfu/mL) of the tested bacterial strain. Then, 1 mL of the tested suspension sample was collected every 1 h for viable cell counting in MHA plate followed by incubation at 37 °C for 24 h.
Lysis of gram-negative spheroplasts
Spheroplasts are gram-negative bacteria in which nearly all of the outer membrane (OM) has been removed. Lysozyme destroys the peptide bonds in peptidoglycan and weakens the cell wall. E. coli was grown in 10 mL of MHB, incubated overnight at 37 °C on a shaker at 180 rpm. Each culture (100 µL) was used to inoculate 20 mL of fresh media and incubated at 37 °C for 2 h at 180 rpm. Spheroplasts were prepared as described by Kikuchi et al. (2015).
Re-suspended spheroplasts and whole cells suspension were adjusted to OD 570 nm of 0.2 and 100 µL of each was added to a clear, flat-bottomed microtiter plate in duplicate wells. Ten microliters of YD1 in 10 mM Tris–HCl buffer (pH 7) was added to test wells for both preparations (spheroplasts and whole cells) at the indicated different final concentrations. To the control, 10 µL of 10 mM Tris–HCl buffer (pH 7) was added in lieu of YD1. The percentage of intact spheroplasts or whole cells was calculated as:
$$= \left( {\frac{\text{Sample OD at time X}}{{{\text{Sample OD at time }}0}} } \right) \times {{100}}$$
Decrease in the OD of the suspension after addition of a membrane-active agent indicates lysis of spheroplasts.
DNA binding assay
The plasmid DNA (150 ng) of E. coli was incubated with increasing concentration of peptides in 20 µL of binding buffer [5% glycerol, 10 mM Tris–HCl (pH 7), 1 mM EDTA, 1 mM DTT, 20 mM KCl, and 50 µg/mL BSA]. The reaction mixtures were kept at room temperature for 30 min, followed by addition of 4 µL of native loading buffer. An aliquot of 12 µL was applied to a 1% agarose gel, and electrophoresis was performed in 0.5 X tris–borate-EDTA buffer.
Transmission electron microscopy (TEM)
TEM analysis was performed according to the method described by Lee et al. (2013). Ten milliliters of 106 cfu/mL E. coli suspension was exposed to 5× MIC (40 µg/mL) of YD1 to observe morphological changes and calculate the percentage killing of E. coli cells.