Development of novel antimicrobial peptides derived from anti-lipopolysaccharide factor of the swimming crab, Portunus trituberculatus.

Anti-lipopolysaccharide factors (ALFs) are a representative host defense protein in crustaceans. In this study, we successfully developed two novel antimicrobial peptides (AMPs), named crab-ALF2A and crab-ALF6A, which contain changes to the amino acid sequences of the lipopolysaccharide binding domain and signal peptide, respectively, of the ALF of the swimming crab Portunus trituberculatus. The crab-ALF2A peptide showed potent antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentration [MEC] 1.51-1.93 μg/mL) and the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (MEC 1.87-1.98 μg/mL), with maximal bactericidal activity at a peptide concentration of 5 μg/mL. The crab-ALF6A peptide also showed potent antimicrobial activity against B. cereus, S. aureus, and S. iniae (MEC 1.49-2.3 μg/mL) and P. aeruginosa and E. coli (MEC 1.72-1.19 μg/mL) at a peptide concentration of 5 μg/mL. Notably, the crab-ALF2A and crab-ALF6A peptides exhibited strong activity against Candida albicans (MECs of 2.11 and 1.95 μg/mL, respectively). These activities were stable following heat treatment. Moreover, the effect of crab-ALF2A and crab-ALF6A peptide treatment on microbe cell morphology was confirmed by scanning electron microscopy. Membrane disruption and damage, and the leakage of cytoplasmic content were clearly observed. A downsizing peptide approach illustrated that the hexapeptide ALF6A8 (RVLLRL) was the shortest peptide showing significant antimicrobial activity. Our approach allows for the generation of novel antimicrobial peptides in a cost effective manner as potential next-generation antibiotics.