Proteomic insights into the multi-target mechanism and therapeutic application of citronellol-loaded carboxymethyl chitosan-based hydrogel for wound infection treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) continues to challenge wound care due to its strong biofilm-forming ability and resistance to conventional antibiotics. In this study, we developed a novel multifunctional hydrogel by incorporating citronellol, a bioactive monoterpenoid, into a carboxymethyl chitosan-hyaluronic acid (CMCS-HA) matrix, yielding a biocompatible platform with potent anti-MRSA activity. Unlike many previous phytocompound-based hydrogels, our work provides in-depth mechanistic insights into citronellol's antibacterial and antivirulence actions through proteomics coupled with STRING network analysis. Citronellol effectively inhibited MRSA growth, disrupted biofilms, and suppressed key virulence factors, including hemolysin, lipase, and staphyloxanthin. Proteomic analysis revealed that citronellol disrupted MRSA virulence by downregulating key adhesins (ClfA, ClfB, FnbA/B, Spa), exotoxins (Psmα4, LukS-PV, Sea), biofilm (IcaA/D, SarA/Z), and quorum sensing (AgrA/C, LuxS) associated proteins in MRSA. These multifactorial effects highlight its potent antibacterial and antivirulence action. The CMCS-HA-citronellol hydrogel demonstrated superior mechanical integrity, cytocompatibility, and sustained drug release. In a zebrafish wound infection model, the hydrogel accelerated wound healing, reduced bacterial burden, and modulated inflammatory and remodeling markers, indicated by decreased IL-1β, MMP-9/13 and elevated TGF-β expression. The combined mechanistic and therapeutic evidence supports the application of citronellol-functionalized hydrogels as a next-generation topical intervention against multidrug-resistant MRSA infections.