Abstract
Big defensins are a family of antimicrobial peptides (AMPs) widespread in molluscs, characterized by a unique structure comprising a β-defensin-like C-terminal domain linked to a hydrophobic N-terminal domain with no homology to known proteins. In the oyster Crassostrea gigas, seven genes encoding Big defensins have been identified. However, the role of Big defensin sequence diversification remains to be elucidated from a functional point of view. Recent studies have revealed a potential function for Big defensins in oysters in the context of the Pacific Oyster Mortality Syndrome (POMS), a polymicrobial disease that has been affecting farms since 2008. The observed inhibition of the expression of certain Cg-BigDefs in oysters susceptible to the disease has indeed suggested that Cg-BigDefs could play a role in controlling the microbiota. My thesis work focused on (1) studying the functional diversification of Cg-BigDefs, and in particular their role in controlling the microbiota of C. gigas, and (2) investigating the mechanism of action of Cg-BigDef1. Thanks to the total synthesis of Cg-BigDef1 and Cg-BigDef5 and the establishment of a collection of culturable bacteria from the microbiota of C. gigas, we have been able to show that the sequence diversification of Cg-BigDefs has broadened the spectrum of activity of this family of AMPs. This is evidenced by a diversification of targets (susceptible bacterial strains) and synergistic activities between variants of the Cg-BigDefs family. The fine modulation of oyster microbiota by Cg-BigDefs was verified in vivo by 16S metabarcoding for Cg-BigDef1. The search for molecular targets of Cg-BigDef1 was undertaken in Staphylococcus aureus. Using complementary and innovative methods (click chemistry, high-resolution microscopy and isogonic mutants), we have shown that teichoic acids (key cell wall polymers) from S. aureus play a decisive role in the antibacterial activity of Cg-BigDef1. The interaction with teichoic acids involves the N-terminal domain of Cg-BigDef1, which brings the C-terminal domain into contact with the bacterial wall. These various results provide a better understanding of the structural diversity of Big defensins from a functional point of view, and provide information on their role in shaping the oyster microbiota and the mechanisms of action underlying their antibacterial activities.