Riluzole attenuates staphyloxanthin biosynthesis and biofilm formation in Staphylococcus aureus.

The rapid emergence and dissemination of antibiotic resistance in Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), pose a major global health challenge and call for alternative therapeutic strategies. S. aureus uses staphyloxanthin (STX) and biofilm as complementary virulence-associated traits that promote immune evasion and persistence at infection sites. STX protects bacteria from reactive oxygen species, while biofilms provide a structured barrier that enhances tolerance to host and antimicrobial stress. Targeting these traits may offer a non-bactericidal strategy for infection control. Here, we identified riluzole as an inhibitor of carotenoid production at non-growth-inhibitory concentrations (IC50 = 34.76 μg/mL) and enhances the susceptibility of S. aureus to immune cell killing. Mechanistically, riluzole interacted with CrtN (KD = 48.6 μM) and reduced its enzymatic activity, while transcriptomic analyses revealed broader transcriptional remodeling associated with reduced expression of several STX biosynthetic genes. In addition, riluzole induced membrane-associated physiological stress, including increased membrane permeability, altered membrane fluidity, ATP depletion, enhanced colistin susceptibility, and inhibition of biofilm formation. Riluzole also attenuated Pseudomonas aeruginosa-associated stimulation of S. aureus pigmentation and oxidative-stress protection. In a murine skin infection model, riluzole promoted bacterial clearance and was associated with improved wound repair. Collectively, these findings substantiate the anti-virulence properties of riluzole against S. aureus, and provide a rationale for its further development as a novel therapeutic strategy for MRSA infections.