A novel type I Crustin from Exopalaemon carinicauda: Antimicrobial ability related to conserved cysteine

Abstract

Crustins are a kind of antibacterial peptides (AMP) existing in crustaceans, and their antibacterial abilities are considered to be related to the conserved WAP domain. In this study, a novel type I Crustin gene was identified in Exopalaemon carinicauda, named EcCru. The deduced amino acid sequence revealed that the conserved cysteine at position 7 in the WAP domain was replaced by aspartic acid. The gene is 405 bp in length, encoding 134 amino acids, and is mainly distributed in gills and hepatopancreas. After Vibrio parahaemolyticus and Aeromonas hydrophila stimulation, the expression of EcCru was significantly up-regulated within 12 h, and then returned to normal levels. The recombinant protein was obtained using the Pichia pastoris expression system, and the recombinant protein had neither antibacterial activity against gram-positive or gram-negative bacteria. But the antibacterial ability emerged when Asp101 was mutated to Cys. Notably, we also obtained a mutant that had a deletion at the 6 th conserved Cys in the WAP domain, and this mutant had antibacterial ability against gram-positive bacteria Bacillus subtilis and B. cereus. This indicates that the conserved cysteine with different positions in WAP domain can have different effects on the antibacterial ability of Crustins.