Teichoic Acids Play a Key Role in the Antibacterial Activity of Big Defensins against Staphylococcus aureus

Crassostrea gigas big defensins (Cg-BigDefs) are a family of two-domain antimicrobial peptides with broad antibacterial activity. The C-terminal domain of Cg-BigDefs harbors a β-defensin-like structure, whereas the ancestral N-terminal domain adopts a globular structure. Here, we developed molecular tools to track the fine interactions of these two domains with Staphylococcus aureus and to gain insight into Cg-BigDef1 mechanisms of action. By using super-resolution microscopy and S. aureus mutants with specific deletions of cell wall components, we found that teichoic acids (TAs) play a key role in the Cg-BigDef1 interaction with S. aureus. A ΔtagO S. aureus mutant lacking cell wall teichoic acids (WTAs) exhibited increased resistance to Cg-BigDef1. Consistently, the binding of Cg-BigDef1 to S. aureus cell wall was significantly reduced in the ΔtagO mutant. In contrast, a ΔdltA S. aureus mutant unable to transfer d-alanine onto lipoteichoic acid (LTA) showed increased susceptibility to Cg-BigDef1 and lysed rapidly in contact with the peptide. Cg-BigDef1 bound to S. aureus cell wall. In addition, competitive binding with exogenously added LTA was sufficient to impair Cg-BigDef1 antimicrobial activity against S. aureus. These data suggest that TAs are conserved molecular motifs recognized by Cg-BigDef1. Finally, we found that Cg-BigDef1 interaction with S. aureus was mediated by its N-terminal domain, which enables the C-terminal β-defensin-like domain to interact with the bacterial cell wall. Altogether, our results identify TAs as important targets for Cg-BigDef1. This interaction appears to be mediated by the ancestral N-terminal domain characteristic of this peptide family.