A novel modified peptide derived from tilapia piscidin 4 with improved cytotoxicity, stability and antibacterial activity against fish pathogens and its underlying antibacterial mechanism.

Tilapia piscidin 4 (TP4) is an amphiphilic cationic antimicrobial peptide derived from Nile tilapia (Oreochromis niloticus), known for its broad-spectrum antimicrobial activity, potent anti-tumor effects, and immunomodulatory property. However, its significant toxicity and poor stability pose major challenges for practical applications. In this study, the TP4 sequence was modified by deleting nine amino acids from the N-terminal region and substituting glycine at the 13th position with cysteine, resulting in a modified peptide designated TP4-16G4C (FSACKAIHRLIRRRRR). The dimer of TP4-16G4C (bis-TP4-16G4C) was obtained by facilitating the formation of disulfide bonds through the oxidation of cysteine. Subsequently, their antibacterial activity, cytotoxicity, stability, and underlying mechanisms were investigated. TP4-16G4C and its dimer exhibited excellent antibacterial activity against a range of fish pathogens, particularly the dimer in vivo. Further study indicated that bis-TP4-16G4C exhibited significantly reduced toxicity toward fish red blood cells and other cell lines, alongside improved stability against proteases and serum, compared to the parental peptide TP4. Mechanistically, bis-TP4-16G4C disrupted the integrity of the bacterial membrane, leading to the leakage of cellular contents; additionally, it interacted with lipopolysaccharides, bound to bacterial genomic DNA, and effectively inhibited bacterial biofilm formation, similar to the action of TP4. In summary, the modified and dimerized antimicrobial peptide bis-TP4-16G4C exhibits reduced toxicity, enhanced stability, and superior antimicrobial activity in vivo, suggesting its greater suitability for practical applications in aquaculture and other fields compared to its parental peptide.