The rising threat of antibiotic-resistant Staphylococcus aureus necessitates novel therapeutic strategies. This study elucidates the dual mechanism by which the NSAID flufenamic acid (FFA) enhances bacterial killing by immune cells. We identified the quorum-sensing AgrAC two-component system as a key target of FFA in S. aureus. Through promoter-reporter assays, EMSA, and mutagenesis, FFA was shown to bind the response regulator AgrA at a novel site (E27) within its regulatory domain, inhibiting virulence gene expression (e.g., α-toxin). This makes FFA the first reported inhibitor targeting this AgrA domain, distinct from existing DNA-binding domain inhibitors.

Concurrently, FFA’s immunomodulatory action is essential for its efficacy. In macrophages, FFA inhibited the NLRP3 inflammasome, a process critical for promoting phagosome-mitochondria colocalization and subsequent bactericidal reactive oxygen species (ROS) generation. This NLRP3-dependent mechanism was validated in vivo, where FFA lost its effect in nlrp3-KO mice. While exhibiting minimal direct antibacterial activity (MIC >400 µM), FFA acted synergistically with gentamicin, significantly reducing bacterial loads in vivo.

Thus, FFA represents a promising anti-virulence agent that uniquely combines suppression of bacterial pathogenicity via AgrA inhibition with potentiation of host innate immunity via NLRP3 blockade. This dual, host-pathogen targeting strategy minimizes selective pressure for resistance and supports the therapeutic potential of repurposing FFA, particularly in topical or combination regimens, against staphylococcal infections.