The combination of hexanal and geraniol in sublethal concentrations synergistically inhibits quorum sensing in Pseudomonas fluorescens-In vitro and in silico approaches.

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Hexanal and geraniol are essential oil components with anti-quorum sensing (QS) activity against Pseudomonas fluorescens. This study demonstrated that QS inhibition (QSI) efficacy of the hexanal and geraniol combination (HG) was significantly higher when compared to those of their mono-counterparts at the same concentration. Tests on P. fluorescens motility, biofilm formation, acyl-homoserine lactones' (AHLs) production, gene expression in vitro, and molecular docking in silico were conducted to evaluate the synergistic effect of hexanal and geraniol on QSI. HG mixture at 0.5 minimal inhibitory concentration (MIC) showed a strong synergistic inhibition of biofilm formation (51.8%), motility (60.13%), and extracellular protease activity (58.9%) of P. fluorescens. The synthesis of AHLs, e.g., C8 -HSL and C12 -HSL, was inhibited by hexanal, geraniol, and HG; both AHLs are responsible for regulating virulence factors in P. fluorescens. The expression of pcoI and gacA genes regulating AHL synthetase and sensor kinase was significantly down-regulated by HG (0.29 and 0.38-fold) at 0.5 MIC. Hexanal and HG showed significant inhibition of the expression of pcoR and gacS genes, which are responsible for AHL receptor protein and response regulation; however, geraniol failed to downregulate the two genes. Molecular docking in silico also supported these findings. Hexanal, which gets inserted into the minor groove of pcoI/pcoR DNA fragments, inhibits the expression of both the genes. Both hexanal (-31.487kcal/mol) and geraniol (-25.716kcal/mol) had a higher binding affinity with PcoI protein than the halogenated furanone C30 (-24.829kcal/mol), which is a known competitor of AHLs. Similarly, hexanal and geraniol strongly bind to the PcoR protein also. It was found that HG at 0.5 MIC could effectively inhibit QS by suppressing the expression of pcoR/gacS and gacA/gacS genes and therefore, could inhibit the motility and biofilm formation of P. fluorescens. The present study indicated that HG at sub-MIC as QS inhibitor could be further developed as a new preservative of agri-food products.