Naturally Occurring 2-Methoxy-1,4-naphthoquinone Attenuates Biofilm Formation and Quorum Sensing-Regulated Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa employs quorum sensing (QS) to coordinate biofilm formation and virulence factor expression, positioning QS as a promising target for anti-infective interventions. This study evaluated the antibiofilm and antivirulence efficacy of 2-methoxy-1,4-naphthoquinone (MNQ), a natural naphthoquinone, against P. aeruginosa PA14. At concentrations of 31.25, 62.5, and 125 μg/mL, which had minimal impact on growth, MNQ suppressed biofilm formation (up to 70% at 125 μg/mL), reduced extracellular polymeric substance production, and inhibited quorum sensing-related signalling activity (85.9%), pyocyanin synthesis (78%), as well as swimming and twitching motilities. Notably, MNQ also exhibited antioxidant properties and inhibited efflux pump activity, suggesting a multifaceted mode of action. Mechanistically, qRT-PCR analysis following treatment with MNQ at 125 μg/mL demonstrated significant downregulation of QS regulatory genes (lasR, lasI, rhlR, rhlI, rhlA) and downstream virulence determinants, corroborating the phenotypic observations. Furthermore, MNQ acted synergistically with azithromycin, cefepime, and ciprofloxacin. Collectively, these findings establish MNQ as a potent QS-directed inhibitor with broad antivirulence activity, underscoring its potential as a lead compound for novel antibiofilm therapeutic strategies.