Abstract
Antimicrobial peptides (AMPs) are ancient innate immune components. Big defensins is a family of AMPs found in a restricted number of animal phyla, in particular mollusks where they have highly diversified. Big defensins are composed of a highly hydrophobic N-terminal region and a C-terminal β-defensin-like region, stabilized by three disulfide bridges. They have been shown to be active against both Gram-positive, Gram-negative bacteria and fungi. Antimicrobial aggregates called nanonets entrapping bacteria have been recently described as the mechanism of action of the Cg-BigDef1 from the oyster Crassostrea gigas. Specifically, the N-terminal domain of Cg-BigDef1 was identified as responsible of nanonet formation. In order to determine whether nanonets are specific to oyster Cg-BigDef1 or common to other big defensins outside this species, we assessed the potential entrapping of bacteria through nanonets of the big defensin from the scallop Argopecten purpuratus, namely ApBD1. Recombinant ApBD1 was produced as a fusion polypeptide which carried a N-terminal His6 tag, with a thrombin cleavage site before the mature peptide sequence and an unfolded C-terminal domain by mutating the last Cys to Arg. Activity of rApBD1 was assessed against the gram-positive bacteria Staphylococcus aureus SG511. rApBD1 inhibited bacterial growth. Moreover, strong immune staining of rApBD1 in numerous areas surrounding bacteria was observed. Overall, results suggest that rApBD1 entrap bacteria in peptide aggregates similar to those reported to Cg-BigDef1. This study demonstrates the conservation of nanonet formation across big defensins and supports further a role for the N-terminal domain in this conserved process.
