绿原酸通过c-di-GMP信号通路调控铜绿假单胞菌生物被膜感染的作用及机制研究

Pseudomonas aeruginosa (P.a) is one of the most common opportunistic pathogens in nosocomial infection. Increasing resistance to antibiotics and host immune defense will occur once the planktonic P.a cells aggregate into biofilms (BFs). Cyclic-di-GMP(c-di-GMP) signaling is konwn as a special second messenger, positively regulates the formation of P.a BF and negatively inhibits the motilities. In the previous study, we had demonstrated that chlorogenic acid could inhibit P.a BF formation and prompt its dispersion in vitro and in vivo. In the preliminary experiment, we also found that chlorogenic acid could enhanced the swimming, swarming and twitching motility of P.a. On this basis, we construct different laboratory and clinical strains with high or low intracellular c-di-GMP levels and label them with green fluorescent protein by gene engineering technique. Then these strains are used to establish various of BF models (static culture, flow chamber system and pulmonary infection in rat models) and various methods (confocal laser scanning microscopy, electronic scanning microscopy, fluorescence in situ hybridization, exopolysaccharides visualization, crystal violet assay and series dilution method of plate counting) are also applied in the present study, aiming to explore and elucidate the effect and relative mechanism of c-di-GMP signaling on the process of P.a BF formation and dispersion, antibiotic tolerance, motility, cell adhesion ability, bacterial load, host inflammatory response and histopathological alteration in the lesion of rat pulmonary P.a BF infection after chlorogenic acid treatment.

铜绿假单胞菌(P.a)是院内感染常见的机会致病菌，一旦其形成生物被膜(BF)可加重病原菌耐药性及对宿主免疫系统的抵抗性。环二鸟苷酸(c-di-GMP)是细菌特有的新型第二信使，c-di-GMP信号通路激活可促进P.a BF的形成和并抑制菌株的运动能力。我们的前期研究已证实金银花活性成分绿原酸可在体内外抑制和破坏P.a BF，并通过预实验发现绿原酸可促进P.a泳动、群集和蹭动。本项目拟在此基础之上，运用基因工程技术构建绿色荧光蛋白标记过表达及低表达c-di-GMP的临床及实验室菌株，并建立体外静止、Flow chamber流动及大鼠肺部P.a BF感染模型，运用形态学定性与定量手段相结合的研究方法，深入探讨绿原酸干预体内外P.a BF形成、抗生素耐受性、运动及细胞黏附能力、肺部BF感染灶细菌学、组织病理与炎症类型改变的过程中，c-di-GMP信号通路的调控作用及所涉及的具体基因与分子机制。

铜绿假单胞菌(P.a)生物被膜(BF)引起的感染具有难治性、反复性、高耐药性及高病死率等特点，该特点与P.a BF形成密切相关，体外研究证实，P.a BF形成受c-di-GMP信号通路调控。本项目在成功构建可实现对胞内c-di-GMP表达量进行动态定量监测的P.a菌株基础上，利用体外静止、follow chamber及动物体内感染模型证实：1）绿原酸可以通过c-di-GMP信号通路调控菌株泳动、群集及运动能力、细胞粘附能力及致病力；2）绿原酸在体外可以浓度及时间依赖性方式抑制P.a BF形成，增强左氧氟沙星、美罗培南及头孢他啶等抗菌素对P.a BF的杀伤作用。3）绿原酸在体外清除P.a BF的分子机制与调控c-di-GMP信号通路关键基因PleQ、WspR和PilZ相关。4）成功构建不同c-di-GMP表达菌株大鼠肺部P.a BF感染模型，证实绿原酸可显著降低P.a BF感染鼠肺部感染灶菌负荷，减轻局部组织炎症病理及免疫损伤，促进P.a BF感染清除，且该作用效果与菌株c-di-GMP信号通路状态相关。项目研究成果拓展了中药金银花活性成分绿原酸的药理作用范围，证实了c-di-GMP信号通路可作为抗P.a BF感染潜在作用靶位之一，并为新型抗P.a BF感染药物研发待定理论依据。