In-silico analysis tools
The first step for in silico analysis was to search in the Antimicrobial Peptide Database (APD) (http://aps.unmc.edu/AP/main.php) for sequences with high similarities to the target bacteriocin. EntPBLAST sequence homology searches using the NCBI protein BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) were performed to identify the template AMPs. To analyze nucleotide sequences, determine the correct nucleotide sequence from protein, and design restriction enzyme digestion sites for cloning, CLC Genomics Workbench Version 3.6.5 software (Redwood City, CA, USA) was used. Then the I-TASSER simulations (Roy et al. 2010; Yang et al. 2015), were conducted to generate structure predictions for EntP, where homologous templates were excluded from the threading template library. I-TASSER simulations predicted the B-factor profile (BFP), the 3D model, and the estimated global accuracy for EntP.
Strains and vectors
Escherichia (E.) coli DH5α cells were grown in LB broth (Sigma Chemical Co., St. Louis, MO, USA) at 37 °C with shaking. CHO cells, pcDNA™3.1(+) vector, BamHI and HindIII restriction enzymes, and kits for plasmid extraction were purchased from Thermo Fisher Scientific (USA).
Construction of the expression vector and transformation
To increase recombinant EntP expression, a His tag was generated by GenScript’s OptimumGene software (Genscript®, USA) and then chemically synthesized by Generay Biotech (Shanghai, China), according to the codon usage of CHO cells. The optimized gene construct encoding a 44-mer EntP peptide plus a 6-His tail was ligated into the multiple cloning site of the plasmid pcDNA3.1(+) with BamHI and HindIII restriction sites at the 5′ and 3′ ends, respectively (GenScript, USA). Ligations were performed with T4 DNA ligase (Roche Molecular Biochemicals, Mannheim, Germany). After verification of the correct nucleotide sequence, E. coli DH5α cells were transformed with the construct using CaCl2 and grown on LB agar contain-ing 100 μg/ml of ampicillin. Colony-PCR was performed to identify colonies containing the recombinant vec-tor. The recombinant vector, named pcDNA3.1(+)-EntP, was extracted using a plasmid extraction kit (Thermo Fisher Scientific, USA) and used to transform CHO cells.
Cell culture and transfection
CHO cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 8–12 mM l-glutamine with 100 μg/ml penicillin/streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen, USA), at 37 °C with 5% CO2 and a relative humidity of 95%. The culture media was renewed every third day and cultures were passaged every 4–5 days. Viable cells were counted using 0.5% trypan blue (Sigma Aldrich, USA). Approximately 2.5 × 105 CHO cells were seeded into 35 mm wells and cultured in DMEM until 60–80% confluent before transfection. CHO cells were transiently transfected with 10 μg of pcDNA3.1(+)-EntP in serum-free media using Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer’s protocol. Seventy-two hours after transfection the medium was replaced with selective medium containing 400 μg ml−1 of neomycin (Sigma Aldrich, USA) to kill untransfected cells. The surviving transformed cells were collected and stored at 4 °C.
Protein production and purification
After establishing the stable recombinant CHO cells and providing an efficient environment for protein production, the supernatants were collected for protein purification by affinity chromatography. Briefly, the supernatant was adjusted to pH 7.0 by the addition of 1 M sodium phosphate buffer, pH 7.0, and filtered through a 0.45 μm membrane filter (Sartorius Stedim, Germany). A HiTrap column was washed with binding buffer containing 20 mM sodium phosphate, pH 7.0, and then the filtered supernatant was loaded onto the column and chromatographed at a flow rate of 3 ml/min. The column was again washed with binding buffer and the bound proteins were eluted with 100 mM sodium citrate buffer, pH 4.6, and collected in Eppendorf microtubes containing neutralization buffer composed of 1 M Tris–HCl, pH 9. The recombinant EntP peptide containing a 6-His tail was purified with Ni–NTA agarose (QIAGEN, USA). The column was washed with distilled water and equilibrated by passing lysis buffer containing 50 mM potassium phosphate, pH 7.8, 400 mM NaCl, 100 m MKCl, 10% glycerol, and 0.5% Triton X-100 through the Ni–NTA agarose. The supernatant was loaded onto the column and chromatographed at a flow rate of 1 ml/min. The column was washed with lysis buffer containing 10 mM and then 30 mM imidazole, and the proteins were eluted with lysis buffer containing 500 mM imidazole and collected in individual microtubes. Finally, a Vivaspin 20 ultrafiltration spin column (Sartorius Stedim, Germany) was used to concentrate and desalt the eluted protein fractions. The EntP peptide concentration was determined by the Bradford method (Bio-Rad Protein Assay) according to the manufacturer’s instructions, using BSA (Protein Assay Standard II) as the standard.
SDS–PAGE and Western blot analysis
The protein samples were electrophoresed by 15% SDS–PAGE under reducing and non-reducing conditions and the gels were stained with Coomassie Brilliant Blue (Merck, Germany). For Western blotting, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 2% BSA overnight at 4 °C. To identify the recombinant EntP peptide the membranes were incubated for 60 min at room temperature with horseradish peroxidase (HRP)-labelled antibody (Santa Cruz, USA). Finally, the recombinant peptide was visualized by enhanced chemiluminescence detection system (ECL) method according to the manufacturer’s instructions (Amersham Biosciences).
Bacterial inhibitory activity of bacteriocin EntP
The minimal inhibitory concentration (MIC) of the purified EntP peptide was evaluated using the broth micro-dilution assay according to the Clinical and Laboratory Standards Institute (Wayne PA, CLSI. 2017). Fourteen bacterial strains were used in the study. Six were clinical isolates including Salmonella (S.) typhi, L. monocytogenes, S. paratyphi C, Shigella dysenteriae e, vancomycin-resistant enterococci (VRE), and carbapenem-resistant Pseudomonas (P.) aeruginosa (CRSA). The other eight bacterial strains were obtained from the ATCC and include the following: S. aureus ATCC-25923, S. aureus ATCC29213, Enterococcus (E.) faecalis ATCC29212, E. coli ATCC25922, P. aeruginosa ATCC27853, methicillin-resistant S. aureus ATCC33591, Acinetobacter (A.) baumannii ATCC13304, and Klebsiella (K.) pneumonia ATCC 700603. After 18–24 h of incubation on Mueller–Hinton agar at 37 °C, a single colony from each strain was transferred into Mueller–Hinton broth (MHB) (HiMedia, India) and adjusted to an optical density (OD) of 0.5 McFarland units. The cultures were diluted in fresh MHB to a final concentration of approximately 5 × 105 colony-forming units (CFUs)/ml. Assays were conducted in 96-well microtiter plates at 10 different concentrations of EntP peptide prepared by two-fold dilutions from 0.5 to 512 μg/ml in MHB. Vancomycin and gentamicin were used as positive references to determine the sensitivity of each bacterial species tested. The MIC was determined as the lowest concentration of EntP peptide that inhibited visible growth after overnight incubation at 37 °C. All tests were performed in triplicate.
Effect of salt, and 50% human plasma on antimicrobial activity
To investigate the activities of the EntP peptide in the presence of high salt concentrations, the MIC was determined as described above, except that fixed concentration of NaCl was added to each well of the microtiter plate. Overnight cultures of S. aureus (ATCC 25923) and E. coli (ATCC 25922) as Gram-positive and Gram-negative model strains were incubated in MHB with 0, 50, 100, or 150 mM NaCl for 4 h. Bacteria were serially diluted and plated in triplicate on Trypticase soy agar (HiMedia, India) plates. CFUs were counted after 24 h of incubation at 37 °C. The stability of EntP peptide in 50% human plasma was evaluated as previously described (Hou et al. 2011). The human plasma was determined to contain no antimicrobial activity before the test. Then 800 μg/ml of recombinant EntP peptide was diluted 1:1 with fresh human plasma and incubated at 37 °C for 0, 3, or 6 h. After incubation, the antimicrobial activity of each sample was determined by MIC assays with S. aureus (ATCC 25923) and E. coli (ATCC 25922).