Identification and characteristics of a novel cecropin from the armyworm, Mythimna separata

Abstract

Background: The recent emergence of antibiotic-resistant strains of bacteria has increased the need to develop effective alternatives to antibiotics. Antimicrobial peptides have been considered as a promising product with several advantages.Results: In this present study, we identified a novel cecropin from the armyworm, Mythimna separata (armyworm cecropin 1, AC-1) by transcriptome sequencing and multi-sequence alignment analysis. The AC-1 precursor comprised 63 amino acid residues, containing a conserved cleavage site of the signal peptide, Ala23-Pro24, while the mature AC-1 included 39 amino acid residues. Chemically synthesized AC-1 exhibited low hemolytic activity against chicken red blood cells, low cytotoxicity against swine testis cells, and effective antimicrobial activity against Salmonella, Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa. Its antimicrobial activity against Salmonella remained after incubation for 1 h at 100 °C or in 250 mM NaCl, KCl, or MgCl2 solution, implying good thermal- and salt-resistant stabilities. The bactericidal effect of AC-1 on E. coli gradually increased with increasing AC-1 concentration, resulting in deformation, severe edema, cytolysis, cell membrane damage, and reducing intracellular electron density. Additionally, recombinant AC-1 protein expressed in E. coli was digested by enterokinase protease to obtain AC-1, which showed similar antimicrobial activity against E. coli to chemically synthesized AC-1.Conclusions: This study identified a novel antimicrobial peptide that may represent a potential alternative to antibiotics.

The antimicrobial effect of a novel peptide LL-1 on Escherichia coli by increasing membrane permeability

“…Additionally, the hemolytic activity of LL-1 was determined using red blood cells from BALB/c mice. Blood containing anticoagulants collected from mice was washed, cells counted, and diluted as previously reported by our laboratory [25]. The red blood cell suspension was mixed with different concentrations of LL-1 (final concentrations: 50, 100, 150, 200, and 250 μg/mL).…”

“…The cytotoxicity of LL-1 was determined in porcine kidney-15 (PK-15) cells through a CCK-8 cell counting kit (Vazyme, Nanjing, China). PK-15 cells were counted, diluted and inoculated into 96-well cell-culture plates, and then cultured at 37 °C for 24 h as previously reported by our laboratory [25]. Different concentrations of LL-1 were added to the 96-well cell-culture plates at final concentrations of 50, 100, 150, 200, and 250 μg/mL, respectively.…”

“…LL-1 was diluted with phosphate-buffered saline (PBS) to 200 µg/mL. The MIC of LL-1 against E. coli was detected by double dilution method as previously described [25]. Kanamycin (200 µg/ mL) was used as a positive control and PBS was used as a negative control.…”