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

Abstract

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.

When the microbiome shapes the host: immune evolution implications for infectious disease

“…This AMP-microbe specificity may also be found in other systems: in Hydra jellyfish, the AMP NDA-1 is uniquely effective against its microbiome associate Curvibacter in vitro , and Curvibacter specifically fails to colonize NDA-1-expressing tissues [ 42 ]. Oyster big Defensins similarly show drastically different inhibitory concentrations against specific microbiome-relevant bacteria [ 43 ]. Finally, a recent GWAS of Persian domestic cats found that a G49E polymorphism in the AMP Calprotectin was a major risk factor for development of ringworm fungal skin disease [ 44 ], suggesting that these highly specific and important peptide–microbe associations are not restricted to invertebrates.…”

“…15.5814 Gram-positive bacterium Cg-BigDef1 in vitro activity tested against ecological microbes De San Nicolas et al . [ 43 ] their table 1 cat Microsporum canis fungus calprotectin GWAS, polymorphism Myers et al . [ 44 ] paper topic human atopic dermatitis unknown cause, autoimmune DEFB1 GWAS, polymorphisms common across two studies Prado-Montes de Oca et al .…”

Cross-talk and mutual shaping between the immune system and the microbiota during an oyster's life

“…Instead, most AMP families have expanded and diversified through gene duplication events followed by rapid molecular diversification [ 56 ], as a result of directional selection pressures [ 61 ]. This molecular diversity creates synergy and enlarges the spectrum of antimicrobial activities of oyster AMP families, particularly Cg- Defs and Cg- BigDefs [ 24 , 62 ]. Remarkably, the gene expansion and sequence diversification of Cg- BigDefs has also conferred specificity to members of this peptide family against bacteria belonging to the oyster microbiota [ 24 ].…”

“…Bivalve microbiota may be manipulated to prevent disease in aquaculture [22]. A fine-tuned dialogue between oyster immunity and its microbiota was evidenced from early life to adult stages [23][24][25]. Recently a first insight into microbial community gene functions has been made accessible, either by shotgun metagenomics [11] or metatranscriptomics [26].…”

The antimicrobial peptide database is 20 years old: Recent developments and future directions

“…The growing research on microbiota added a new dimension to AMP research due to its connection with a variety of human diseases (Wang et al, 2023; Wehkamp et al, 2005). AMPs are important players that shape microbiota (de San et al, 2022; Pierre et al, 2023; Snelders et al, 2021). Remarkably, a Paneth cell peptide YY was able to selectively eliminate pathogenic hyphae but not the commensal yeast form of Candida albicans (Pierre et al, 2023).…”