Assessment of furobenzopyran from Ammi visnaga in disrupting Pseudomonas aeruginosa biofilms and suppressing associated virulence factors.

Abstract

Ammi visnaga, an herbal plant containing the furobenzopyran derivative (khellin) with therapeutic effects, remains unexplored for its potential to disrupt the biofilm formation and suppress the virulence factors mediated through the Las quorum-sensing system in Pseudomonas aeruginosa. The current study investigates the efficacy of khellin in inhibiting biofilm formation, suppressing biofilm-associated virulence factors at sub-minimum inhibitory concentration (sub-MIC) levels of ≤9 μg/mL, and its interactions with LasR are evaluated through molecular docking and dynamics simulation. In-silico analysis using the 'aBiofilm' web tool predicted a strong antibiofilm potential for khellin, with no prior reports of such activity. This prediction was supported by confocal laser scanning microscopy, which demonstrated significant biofilm inhibition at 9 μg/mL, along with noticeable microbial distortion. Further assessments showed that sub-MIC levels of khellin effectively reduced biofilm-associated virulence factors, including swimming and swarming motility, rhamnolipid content, cell surface hydrophobicity, alginate, and exopolysaccharide production, in a dose-dependent manner, though the extent of inhibition varied among these factors. Molecular docking analysis yielded a score of -7.285 kcal/mol, indicating a favorable binding, and 'Molecular Mechanics Generalized Born Surface Area' binding free energy of -28.32 kcal/mol confirms a stable and energetically favorable interaction with the target protein. The stability of the khellin-protein complex was validated through a 100 ns molecular dynamics simulation using the 'Optimized Potentials for Liquid Simulations-All Atom' force field model. Results of root mean square deviation, root mean square fluctuation, radius of gyration, intramolecular hydrogen bonds, molecular surface area, solvent accessible surface area, and polar surface area confirmed that khellin maintained stable interactions throughout the simulation. These findings suggest khellin as a potential candidate for treating Pseudomonas-associated biofilm infections and provide strong evidence that khellin may function as a quorum-sensing inhibitor of the P.aeruginosa's LasR protein.