The study shows that a virus‑derived protein called Gp05 helps MRSA bacteria change their membrane fats, making them harder for the body’s natural antibiotic peptide LL‑37 and for drugs like vancomycin to kill. Removing Gp05 flips the membrane back, so the bacteria become more vulnerable to LL‑37, immune cells, and antibiotics.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of endovascular infections. The prophage-encoded protein Gp05 has been identified as a critical virulence factor that contributes to MRSA persistence during vancomycin treatment in an experimental endocarditis model. However, the mechanisms underlying this persistence phenotype remain poorly understood. This study investigated the genetic factors contributing to Gp05-associated MRSA persistence utilizing RNA sequencing (RNA-seq) on an isogenic MRSA strain set, including a clinical persistent bacteremia isolate, its chromosomal gp05 deletion mutant, and gp05-complemented strains. RNA-seq revealed significant downregulation of the GraSR two-component regulatory system and its downstream genes, mprF and dltABCD, in the gp05 deletion mutant compared to the wild-type and gp05-complemented strains. This downregulation led to a substantial shift in membrane phospholipid composition, with an increased phosphatidylglycerol and a corresponding decrease in lysyl-phosphatidylglycerol. These changes resulted in increased susceptibility of the gp05 deletion mutant to human cationic antimicrobial peptide (CAMP) LL-37, neutrophils, and vancomycin. These results were confirmed in an isogenic gp05 overexpression strain set in MRSA JE2 background. Gp05 modulates MRSA surface phospholipid components and charge, offering new insights into the molecular mechanisms underlying Gp05-mediated persistence in endovascular infections and potential therapeutic targets to combat these infections.