槲皮素抑制铜绿假单胞菌生物膜形成的作用机制研究

The treatment of bacterial biofilm infections is a still challenge in clinical. Inhibition of the quorum sensing system (QS) is considered as an effective means for treating the bacterial biofilm infections. Our preliminary research found that quercetin could inhibit and reverse partially the bacterial biofilm formation. And this kind of effects was related with the lower expression of lasI and rhlI in the lasI/lasR and rhlI/rhlR system. It was therefore hypothesized that quercetin inhibited biofilm formation probably through inhibiting the QS of Pseudomonas aeruginosa (PA). In this study, we are aimed to determine the optimized inhibitory concentration and time of quercetin against QS of PA. And in order to confirm that quercetin inhibits the bacterial biofilm formation through the lasI/lasR or (and) rhlI/rhlR systems, we intent to construct the lasI，rhlI and lasI/rhlI genes deletion strains and analyze changes of virulence factors, signaling molecules, biofilm formation and related biofilm regulatory genes of PA. Then, we would pinpoint upstream regulatory proteins under the action of lasI gene inhibition induced by quercetin, using DNA affinity chromatography and mass spectrometry, in order to get the target of quercetin inhibiting biofilm formation. We believe that the results of this study can provide theory basis for reasonable biofilm infection prevention in the practice for clinical anti-infection.

细菌生物膜感染是临床亟待解决的难题，抑制群体感应系统（Quorum sensing system，QS）被认为是解决该问题的关键途径。我们前期研究发现槲皮素能抑制并部分逆转生物膜形成，该作用中lasI/lasR和rhlI/rhlR 群体感应系统lasI和rhlI的表达降低是主要变化。推测其抑制铜绿假单胞菌QS系统可能是抑制生物膜形成的重要机制。本项目拟以铜绿假单胞菌为研究对象，明确槲皮素抑制QS的最佳抑制浓度和时间，并构建lasI基因、rhlI基因以及lasI/rhlI双基因缺失株，分析毒力因子，信号分子，生物膜形成及相关调控基因的变化，确证槲皮素通过lasI/lasR或(和)rhlI/rhlR系统抑制生物膜形成，并利用DNA亲和层析与质谱技术，明确槲皮素对lasI基因主要启动区段调控蛋白的影响，推测槲皮素抑制BF形成的靶标。本课题的实施将为临床采取合理的BF感染防治策略提供理论依据。

细菌生物膜感染是临床亟待解决的难题，我们前期研究发现槲皮素能抑制并部分逆转铜绿假单胞菌生物膜形成，但具体机制有待进一步研究，因此本研究的目的是探索槲皮素抑制铜绿假单胞菌生物膜形成的作用机制。目前已完成项目任务书全部内容，研究结果主要包括如下三个方面：① 槲皮素对铜绿假单胞菌PAO1不具有直接抗菌效应，但其可显著抑制生物膜形成、细菌粘附以及绿脓菌素等毒力因子分泌；② 对比lasI、rhlI和lasI/rhlI基因敲除前后槲皮素药理作用的变化，确证槲皮素通过抑制lasI/lasR和rhlI/rhlR信号系统进而抑制生物膜形成；③对比vfR基因敲除前后槲皮素药理作用的变化，证实vfR是槲皮素抑制lasI和生物膜形成的关键环节。综上，本研究探明了槲皮素抑制铜绿假单胞菌生物膜形成的具体作用机制，即槲皮素通过抑制vfR表达，调控lasI/lasR和rhlI/rhlR信号系统，进而抑制铜绿假单胞菌生物膜形成。本研究的完成深化了我们对铜绿假单胞菌生物膜形成和抑制机制的认识。