Functional Diversification of Oyster Big Defensins Generates Antimicrobial Specificity and Synergy against Members of the Microbiota.

Big defensins are two-domain antimicrobial peptides (AMPs) that have highly diversified in mollusks. Cg -BigDefs are expressed by immune cells in the oyster Crassostrea gigas , and their expression is dampened during the Pacific Oyster Mortality Syndrome (POMS), which evolves toward fatal bacteremia. We evaluated whether Cg -BigDefs contribute to the control of oyster-associated microbial communities. Two Cg -BigDefs that are representative of molecular diversity within the peptide family, namely Cg -BigDef1 and Cg -BigDef5, were characterized by gene cloning and synthesized by solid-phase peptide synthesis and native chemical ligation. Synthetic peptides were tested for antibacterial activity against a collection of culturable bacteria belonging to the oyster microbiota, characterized by 16S sequencing and MALDI Biotyping. We first tested the potential of Cg -BigDefs to control the oyster microbiota by injecting synthetic Cg -BigDef1 into oyster tissues and analyzing microbiota dynamics over 24 h by 16S metabarcoding. Cg -BigDef1 induced a significant shift in oyster microbiota β-diversity after 6 h and 24 h, prompting us to investigate antimicrobial activities in vitro against members of the oyster microbiota. Both Cg -BigDef1 and Cg -BigDef5 were active at a high salt concentration (400 mM NaCl) and showed broad spectra of activity against bacteria associated with C. gigas pathologies. Antimicrobial specificity was observed for both molecules at an intra- and inter-genera level. Remarkably, antimicrobial spectra of Cg -BigDef1 and Cg -BigDef5 were complementary, and peptides acted synergistically. Overall, we found that primary sequence diversification of Cg -BigDefs has generated specificity and synergy and extended the spectrum of activity of this peptide family.