Eugenol exhibits anti-virulence properties by competitively binding to quorum sensing receptors

The primary objective of this study was to ascertain the anti-biofilm and anti-virulence properties of sub-minimum inhibitory concentration (MIC) levels of eugenol against the standard strain PAO1 and two multi-drug resistant P. aeruginosa clinical isolates utilizing quorum sensing inhibition (QSI). Eugenol at 400 μM significantly reduced biofilm formation on urinary catheters and the virulence factors (VF) including extracellular polysaccharides, rhamnolipid, elastase, protease, pyocyanin, and pyoverdine (p < 0.001). Further, eugenol exhibited a marked effect on the production of QS signals (AIs) (p < 0.001) without affecting their chemical integrity. In silico docking studies demonstrated a stable molecular binding between eugenol and QS receptor(s) in comparison with respective AIs. Investigation on reporter strains confirmed the competitive binding of eugenol to a QS receptor (LasR) as the possible QSI mechanism leading to significant repression of QS associated genes besides the VF genes (p < 0.001). This study provides insights, for the first time, into the mechanism of the anti-virulence properties of eugenol.

本研究的首要目标是采用群体感应抑制（quorum sensing inhibition, QSI）策略，探究亚最低抑菌浓度（sub-minimum inhibitory concentration, MIC）的丁香酚对铜绿假单胞菌（Pseudomonas aeruginosa, P. aeruginosa）标准菌株PAO1及两株多重耐药临床分离株的抗生物膜与抗毒力活性。实验结果显示，浓度为400 μM的丁香酚可显著抑制尿导管表面的生物膜形成，并显著降低胞外多糖、鼠李糖脂、弹性蛋白酶、蛋白酶、绿脓菌素与绿脓杆菌素等毒力因子（virulence factors, VF）的产生（p < 0.001）。进一步研究表明，丁香酚可对群体感应信号（autoinducers, AIs）的合成产生显著抑制效果（p < 0.001），且不会破坏其化学结构完整性。计算机虚拟对接（in silico docking）实验结果显示，相较于对应的群体感应自诱导物，丁香酚可与群体感应受体形成稳定的分子结合。针对报告菌株（reporter strains）的实验证实，丁香酚可竞争性结合群体感应受体LasR，这一作用可能是其群体感应抑制的核心机制，可显著抑制群体感应相关基因以及毒力因子相关基因的表达（p < 0.001）。本研究首次揭示了丁香酚抗毒力活性的作用机制，为相关领域提供了全新的研究视角与理论参考。