Lysyl-phosphatidylglycerol promotes cell-to-cell contact and biofilm formation of Staphylococcus aureus as a biofilm matrix component

Abstract

Staphylococcus aureus biofilms contribute significantly to persistent infections and antibiotic resistance, supported by a complex extracellular matrix. While their proteins, polysaccharides, and extracellular DNA have been well studied, the role of phospholipids in biofilm architecture remains underexplored. Here, we identify extracellular phospholipids within the biofilm matrix, particularly lysyl-phosphatidylglycerol (Lys-PG), as critical structural elements in S. aureus biofilms. Bacterial phospholipase A1 (PLA1), which hydrolyzes phospholipid acyl ester bonds, effectively dispersed pre-formed biofilms and prevented biofilm formation by hydrolyzing extracellular phospholipids, without affecting bacterial growth or exhibiting cytotoxicity. Microscopic analyses revealed that PLA1 disrupts membranous nanostructures integral to biofilm stability. Lipidomic analysis demonstrated an enrichment of Lys-PG with specific fatty acid species within the biofilm matrix and confirmed their hydrolysis by PLA1. Mechanistically, Lys-PG promotes bacterial aggregation by acting as a molecular glue through electrostatic and hydrophobic interactions. Deletion of mprF, responsible for Lys-PG synthesis, markedly impaired biofilm formation. These findings uncover a previously unrecognized structural role of extracellular phospholipids in biofilm architecture and suggest that targeting Lys-PG and its biosynthetic pathway represents a promising strategy for biofilm control.