Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 μM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.