Identification of antimicrobial peptide
Third instar larvae of the armyworm,Mythimna separata were ground in liquid nitrogen and total RNA was extracted for transcriptome sequencing. The known AMP gene sequence was selected to align the transcriptome sequence using MegAlign software. The primers were designed on the basis of the armyworm gene acquired by sequence alignment (sense primer: 5′-TTTGAATTAAGAACAAT–3′; antisense primer: 5′-CTATTTTCCTAAAGCTT–3′). The gene was amplified by PCR using the above primers with a Premix LA Taq (Takara, Dalian, China) according to the manufacturer’s instructions. The PCR conditions were as follows: denaturation at 94 °C for 4 min, 36 cycles of denaturation at 94 °C for 40 s, annealing at 57 °C for 35 s, and elongation at 68 °C for 25 s, and a final elongation at 68 °C for 8 min. The PCR-amplified products were cloned into the pMD18-T vector (Takara, Dalian, China) and positive plasmids were sequenced.
Multi-sequence alignment of cecropins from different insects
The amino acid sequence of the AC–1 precursor was derived from the nucleotide sequence and subjected to multi-sequence alignment with the respective cecropins of different insects from the protein database at the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/protein/?term = cecropin) using Vector NTI Advance® 11.5.3 software.
Physicochemical characteristics and structure prediction of AC–1
The physicochemical characteristics of AC–1 were predicted by the ExPASy Bioinformatics Resource Portal (http://www.expasy.org/tools/) and its secondary structure was predicted using a novel online computational framework PEP-FOLD3.5 (http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) [20]. The secondary structure components of AC–1 were calculated using an online SOPMA secondary structure prediction method (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page = npsa _sopma.html).
Hemolytic and cytotoxic activities of AC–1
AC–1 was synthesized by Shanghai Gil Biochemical Co., Ltd., China. Its hemolytic activity was tested using blood drawn from chickens and treated with sodium citrate anticoagulant. The treated blood was centrifuged at 3000x g for 10 min and washed three times in phosphate buffered saline (PBS). The red blood cells were counted and then diluted to 2×107/mL, and 100 μL of chicken red blood cell suspension was mixed with 100 μL of different concentrations of AC–1 (50, 100, 200, 300, 400, and 500 μg/mL). Triton X–100 solution was selected as a positive control and PBS as a negative control. After incubation for 1 h at 37 °C, the mixture was centrifuged at 3000 x g for 10 min and the absorbance of the supernatants was then detected at 405 nm (OD405). The hemolysis ratio was calculated by the formula: hemolysis ratio = (A405peptide-A405PBS)/(A405Triton-A405PBS)×100%.
The cytotoxicity of AC–1 was evaluated by CCK–8 cell counting kit (Vazyme, Nanjing, China) in swine testis (ST) cells as described previously, with minor modifications [21]. A total of 100 μL of cells (about 5×104 /mL) per well was added into 96-well cell-culture plates and then incubated for 24 h at 37 °C. Different concentrations of AC–1 (five dilutions from 100 μg/mL to 500 μg/mL) were added to the cells with further incubation for 12 h at 37 °C, followed by the addition of 10 μL of CCK–8 reagent into each well. The cell-culture plates were incubated for 1 h at 37 °C and the absorbance was determined at 450 nm using an automatic microplate reader. Each experiment was repeated three times.
Antimicrobial assay of AC–1
The antimicrobial activity of AC–1 was analyzed by determining the minimum inhibitory concentration (MIC) against different bacteria, as described previously, with minor modifications [22]. The synthesized AC–1 was dissolved in PBS and added into 96-well microtiter plates at two-fold dilutions. All the bacterial strains were cultured in Luria-Bertani (LB) broth at 37 °C to exponential phase. The bacterial solutions were diluted to 2×106 colony forming units (CFUs)/mL and then added to 96-well microtiter plates at 50 μL per well, and 100 μL of AC–1/bacteria solution was fully mixed and incubated for 16 h at 37 °C. Resazurin (10 μL 6 mM) was then added to each well and incubated for a further 3 h. The color change was observed in each well. Ampicillin and kanamycin were used as positive controls and PBS and LB broth as negative controls. The MIC was recorded as the concentration of the peptide in the last well that remained blue.
Thermal- and salt-resistant stabilities of AC–1
We evaluated the thermal- and salt-resistant stabilities of AC–1 by determining its antimicrobial activities against Salmonella according to the inhibition zone method, after treatment at different temperatures and with different concentrations of NaCl. Salmonella was cultured in LB broth at 37 °C to exponential phase, diluted to 2×109 CFUs/mL, and 100 μL of diluted bacterial solution was then fully mixed with 100 mL of sterilized LB solid medium and poured into a sterile culture dish. After cooling, the culture dish was punched using a diameter-same hole puncher. The treated AC–1 solution was added into each well. Ampicillin was used as a positive control and PBS as a negative control. The culture dishes were incubated at 37 °C for 12 h and the diameters of the inhibition zones were measured using vernier calipers. Each experiment was carried out in triplicates.
Statistical analysis
Data were analyzed using GraphPad Prism 6 software. A value of p < 0.05 was considered significant and p < 0.01 was considered highly significant.