Design of a cyclic peptide targeting intracellular Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major clinical challenge, particularly intracellular MRSA infections are difficult to treat because antimicrobial agents must combine stability, host-cell access and bacterial target engagement. Cyclotides offer highly stable cyclic scaffolds for peptide engineering, but their use as intracellular antimicrobial protein inhibitors remains largely unexplored. Here, we engineered a cyclotide-grafted derivative of the antimicrobial peptide KTR by inserting it into the MCoTI-I scaffold, generating the cyclic construct MCo-KTR2. Molecular docking and molecular dynamics suggested potential interactions between MCo-KTR2 and the resistance-associated penicillin-binding protein PBP2a. Site-directed mutagenesis and fluorescence polarization assays indicated that specific residues contribute to binding in vitro. Although MCo-KTR2 displayed lower activity than linear KTR in standard MIC assays, cyclotide grafting increased serum stability by more than 30-fold and enhanced cellular uptake, colocalising with cytosolic S. aureus during infection. These properties were associated with improved activity against intracellular bacteria without detectable cytotoxicity or haemolytic activity. Furthermore, MCo-KTR2 showed higher antibacterial activity when combined with the membrane-active compound Visomitin as well as in combination with vancomycin and gentamicin. Together, these findings identify cyclotide grafting as a strategy to improve peptide stability and intracellular delivery, and support MCo-KTR2 as a scaffold for further optimization against intracellular MRSA infections.