Cyclic dipeptides inhibit Staphylococcus aureus biofilm formation and virulence via agr-quorum sensing, ica, and sarA...

Staphylococcus aureus is a major human pathogen mainly involved in chronic biofilm-related infections, especially at epithelial surfaces and wound sites, owing to its ability to form antibiotic-resistant biofilms and release virulence factors. Conventional antibiotics often fail to eradicate established biofilms and may contribute to the emergence of drug resistance, underscoring the urgent need for alternative biofilm-inhibiting and antivirulence strategies. Herein, we synthesized several dicyclic peptides, including cyclo(L-Phe, L-Hyp) and its stereoisomers cyclo(L-Phe, D-Hyp) and cyclo(D-Phe, L-Hyp), to evaluate their ability to inhibit S. aureus biofilm formation. All cyclic dipeptides exhibited a minimum inhibitory concentration (MIC) of 1 mg/mL. Biofilm inhibition was assessed via crystal violet staining and confocal laser scanning microscopy. At 0.5 mg/mL (1/2 MIC), the compounds exhibited superior inhibition of 24 h biofilm formation compared with vancomycin, with cyclo(L-Phe, D-Hyp) showing the most profound inhibitory activity. qRT-PCR revealed that at 0.25 mg/mL (1/4 MIC), cyclo(L-Phe, D-Hyp) significantly downregulated the expression of the Agr-quorum-sensing system (RNAIII: -75.7%; hla: -61.7%; psm-α: -73.3%), the ica operon (icaA: -71%; icaD: -76.7%), and sarA (-69.3%) (P < 0.0001). Notably, cyclo(L-Phe, D-Hyp) showed low cytotoxicity (CC₅₀ = 5.13 ± 0.27 mg/mL) and negligible hemolysis (<1 %) at twice its MIC, indicating a favorable safety margin for antimicrobial use. These cyclo dipeptides can be formulated for topical delivery to sites such as skin, mucosa, or open wounds, providing a practical approach for localized treatment of biofilm-associated infections. These findings identify cyclo(L-Phe, D-Hyp) as a promising lead for the development of topical anti-infective agents targeting chronic S. aureus biofilm-associated infections.