铜绿假单胞菌PcrV诱导巨噬细胞M1极化的分子机制及其在清除细菌生物膜中的作用研究

Biofilm formation is a major factor contributing to the chronicity of infection and mortality in Pseudomonas aeruginosa (P. aeruginosa)-infected patients. To date few methods have been reported to be effective in controlling biofilm-associated infection. Polarization of macrophage towards a M1 phenotype helps to trigger host Th1 immune response and subsequent biofilm elimination. Researches have shown that the TLR2-JNK signal pathway is pivotal for skewing macrophage polarity towards M1 phenotype upon bacterial infection. In our previous studies, we found that P. aeruginosa PcrV interacts with TLR2, and the treatment of macrophage with PcrV polarizes macrophage towards a M1 phenotype in accompany with the activation of its JNK signal pathway. Thus, we suppose that PcrV might induce macrophage M1 polarization by activating TLR2-JNK signal pathway, and might also exhibit a potential anti-biofilm efficacy. To verify this hypothesis, we firstly propose to identify the critical binding sites between PcrV and TLR2 by constructing the truncated and site-directed mutants. Secondly, we aim to clarify the mechanism of PcrV in inducing macrophage M1 polarization via TLR2-JNK pathway by observing the expression change of cellular factors when treating the TLR2 deficient or mutated macrophage with the PcrV mutants and JNK inhibitor or activator. Lastly, the anti-biofilm efficacy of M1 macrophage polarized by PcrV will be confirmed in a murine model of subcutaneous catheter-related biofilm infection. This investigation might provide a new approach to treat the P. aeruginosa biofilm-associated infection.

铜绿假单胞菌（PA）生物膜形成是其感染迁延不愈并导致高死亡的重要因素，目前仍无有效控制手段。巨噬细胞M1极化是促进机体Th1免疫应答并清除生物膜的有效方式。研究表明TLR2-JNK通路是巨噬细胞识别病原后发生M1极化的关键通路。课题组预实验发现，PA的PcrV蛋白与TLR2具有相互作用，经PcrV处理的巨噬细胞呈M1极化并伴随JNK通路激活。由此推测PA的PcrV可通过TLR2-JNK通路诱导巨噬细胞M1极化，进而发挥生物膜清除效应。本项目拟采用截短及定点突变技术，明确PcrV与TLR2相互结合的关键位点；采用蛋白突变体及JNK通路拮抗剂、激动剂处理TLR2缺失或突变巨噬细胞，观察细胞因子的表达变化，阐明PcrV通过TLR2-JNK通路诱导巨噬细胞M1极化的机制；建立小鼠皮下导管生物膜模型，明确PcrV诱导巨噬细胞M1极化对PA生物膜清除作用。研究将为PA生物膜感染的临床防控提供新的途径。

铜绿假单胞菌（PA）生物被膜可通过多种策略逃避宿主免疫防御系统以达到长期持续性感染。然而，细菌何种组分参与上述过程目前仍不明确。我们通过构建PA皮下导管生物膜模型，并通过RT-qPCR方法、免疫荧光、CFU定量等方法检测PcrV基因表达及浸润的巨噬极化状态发现，在PA生物被膜感染时，其III型分泌系统针状结构PcrV蛋白的基因表达水平明显下调，而PcrV基因表达水平下调促进PA的免疫逃逸及PA感染组织M2极化巨噬细胞的浸润。采用Escherichia coli表达系统表达的PcrV纯化蛋白处理PA皮下导管生物被膜，通过ELISA和CFU定量等方法检测组织炎症因子、细菌量等发现，PcrV促进生物膜感染组织的炎症应答及生物被膜细菌清除。进一步通过RT-qPCR、流式、Western blotting等实验证实PcrV可促进巨噬细胞M1极化及M1极化诱导的促炎和抗菌细胞因子（如诱导型NO和活性氧）产生和对细菌吞噬。进一步机制研究表明，PcrV诱导巨噬细胞M1极化通过激活MAPKs和NF-κB信号通路。综上结果提示，PcrV具有调节宿主免疫应答以促进PA生物膜感染的作用。此外，上述结果也显示出PcrV在治疗细菌生物被膜感染的临床应用前景。. 在本课题的资助下，已发表SCI论文2篇，中文核心期刊综述1篇；申请国家发明专利1项，培养硕士研究生2名。