Discovery and Characterization of a New Crustin Antimicrobial Peptide from Amphibalanus amphitrite

Crustins are an antimicrobial peptide (AMP) family that plays an important role in innate immunity in crustaceans. It is important to discover new AMPs from natural sources to expand the current database. Here, we identified and characterized a new crustin family member, named AaCrus1, from Amphibalanus amphitrite. AaCrus1 shares high identity (48.10%) with PvCrus, a Type I crustin of Penaeus vannamei that possesses a whey acidic protein (WAP) domain. AaCrus1 contains 237 amino acids and eight cysteine residues forming conserved 'four-disulfide core' structure. Our recombinant AaCrus1 (rAaCrus 1) could inhibit the growth of two Gram-positive bacteria (Staphylococcus aureus, Bacillus sp. T2) and four Gram-negative bacteria (Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio alginolyticus) with a minimum inhibitory concentration of 3.5-28 μM. It can further induce agglutination of both Gram-positive and Gram-negative bacteria. rAaCrus1 can bind to bacteria and damage bacterial cell membranes. Furthermore, rAaCrus1 disrupted biofilm development of S. aureus and V. parahaemolyticus. Our discovery and characterization of this new crustin can be further optimized as a good alternative to antibiotics.