World Aquaculture - June 2023

WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2023 67 AMPs include defensins, drosomycin and protegrin (Matsuzaki et al. 1997). Linear extended AMPs and loop AMPs are the other two classes of AMPs. These peptides are linear in shape without secondary structure, and overexpression of certain amino acids characterizes their sequences. AMPs of this class are rich in histidine, proline or arginine. Examples of linear extended AMPs include indolicidin and apidaecin. Loop AMPs adopt a loop conformation with one disulfide bridge (Seo et al. 2012). Mechanism of Action AMPs can act by two mechanisms: membrane-disruptive and non-membrane disruptive. The membrane-disruptive mechanism is based on the attraction of AMPs toward the cell membrane, attachment of AMPs to the membrane, followed by insertion of AMPs into the membrane, leading to membrane disruption. The non-membrane disruptive mechanism is based on the binding of AMPs to intracellular molecules such as DNA, RNA and enzymes, inhibiting various processes, such as replication, transcription and translation. The membrane-disruptive mechanism of AMPs is based on the electrostatic interaction between the cationic AMPs and the negatively charged bacterial cell membrane. Hydrophobic interactions between hydrophobic residues of AMPs and the bacterial membrane also play a minor role. AMPs attach themselves to the bacterial membrane and acquire a parallel orientation with the membrane. When the AMP concentration increases up to the threshold value, peptides adopt a perpendicular orientation and initiate insertion into the membrane, leading to membrane disruption. Three models have been proposed to describe the membranedisruptive mechanism of AMPs: the barrel-stave model, the carpet model and the toroidal pore model (Fig. 1). In the barrel-stave model, AMPs aggregate as monomers onto the membrane in a perpendicular orientation and adopt a barrel stave structure followed by insertion into the membrane (Yang et al. 2001). In the carpet model, AMPs form high-density clusters at the membrane surface, covering the whole membrane like a carpet. These clusters result in significant curvature strain in the membrane, leading to its disruption (Pouny et al. 1992). The toroidal pore model is based on the combined action of the barrel-stave model and the carpet model. AMPs adsorb onto the membrane in a carpet manner and then insert into the membrane in a perpendicular orientation (Hancock and Chapple 1999). Synthetic and Recombinant AMPs To use AMPs in aquaculture, several approaches have been proposed, including the use of synthetic AMPs and recombinant AMPs. In the case of synthetic AMPs, peptides are designed and synthesized by solid-phase peptide synthesis techniques. These techniques involve coupling amino acids in a specific order, protecting groups and deprotection steps to ensure correct folding and function. The resulting synthetic peptide can be purified using high-performance liquid chromatography (HPLC) techniques and characterized using mass spectrometry and other analytical techniques. Another approach is to use recombinant DNA technology to express the peptide in a heterologous host, such as bacteria or yeast. This involves first identifying and cloning the gene for the desired AMP, then inserting it into the genome of the host organism so it can be expressed and purified. This approach enables production of large quantities of the peptide, making it more cost-effective than chemical synthesis. In either case, the synthetic AMP can be optimized for specific applications by modifying its sequence or structure to enhance its activity against particular pathogens or increase its stability or solubility. This can involve making single-point mutations, incorporating non-natural amino acids, or adding functional groups to the peptide chain. Synthetic AMPs have shown promise in a variety of applications, including as antimicrobial coatings on medical devices, as topical treatments for skin infections and as feed additives to improve animal health and growth in agriculture. As research into AMPs continues, it is likely that their use will expand to additional fields and applications, further underscoring the importance of the ability to produce them synthetically. Advantages of AMPs over Antibiotics as Therapeutic Agents The use of AMPs in aquaculture has several advantages over traditional antimicrobial agents. • Broad-spectrum activity. AMPs can kill or inhibit the growth of a wide range of microorganisms, including bacteria, fungi, viruses and parasites. In contrast, antibiotics are usually effective against a limited number of bacterial species. • Low likelihood of resistance development. AMPs have a unique mode of action that targets multiple cellular pathways, as in the disruption of the pathogen’s cell membrane, making it difficult for microorganisms to develop resistance. In contrast, the widespread use of antibiotics has led to the emergence of antibioticresistant strains of bacteria. • Rapid action. Antimicrobial peptides work quickly to kill microorganisms, usually within minutes to hours. In contrast, antibiotics may take several hours or days to become effective. • Minimal toxicity. Antimicrobial peptides are usually less toxic to host cells than antibiotics. • Potential for use in combination therapies. Antimicrobial peptides can be used in combination with other therapies, including antibiotics, to enhance their efficacy and reduce the development of resistance. FIGURE 1. The mechanism of action of antimicrobial peptides (AMPs). (CONTINUED ON PAGE 68) Pathogen (Virus, Bacteria, Parasite) Attack