单分子荧光成像研究绿脓杆菌信号分子调控宿主细胞信号转导的分子机制

This project aims to develop and apply live-cell single-molecule detection techniques to investigate molecular mechanisms of how a chemical signal molecule, 3-oxo-C12-HSL, secreted by Pseudomonas Aeruginosa (PA) regulates host cell signal transduction and to discuss a new theory of “ligand-independent receptor activation induced by membrane alteration” proposed herein. PA is one of the most important pathogenic bacteria causing post operation infection and an immune compromised individuals. 3-oxo-C12-HSL secreted by PA can inhibit the host immune cells, but its binding receptor has never been found at present. Thus the underlying mechanism is not known. Here a new receptor activation mode is proposed in which incorporation of 3-oxo-C12-HSL molecules into the host membrane alters the structure of lipid-raft, which leads to receptor oligomerization and spontaneous signal initiation. Live-cell single-molecule fluorescence imaging/tracking and super-resolution imaging will be developed to characterize the distribution of 3-oxo-C12-HSL on cells and the effects of 3-oxo-C12-HSL on the arrangement of membrane lipid-molecules and on the stoichiometry and mobility of TNF-α receptor and others. Meanwhile, the structure and function linkage of 3-oxo-C12-HSL and its analogs will be studied as well. The results of this study will complement and improve the existing ligand-mediated receptor activation theory and provide new theoretical and experimental evidence on the clinical control and treatment of infection and the development of new drugs in the future.

本项目旨在发展和应用活细胞单分子检测表征技术，研究绿脓杆菌分泌的化学信号分子3-oxo-C12-HSL调控宿主细胞信号转导的分子机制，探讨“非配体依赖的膜诱导受体激活”新原理。绿脓杆菌是临床上是引发术后感染的重要致病菌，它分泌的3-oxo-C12-HSL具有抑制宿主免疫细胞的功能，但至今未发现它的结合受体，其作用机制未知。本项目提出3-oxo-C12-HSL嵌入宿主细胞膜，改变膜上脂筏结构，从而影响膜上受体多聚化激活的新观点；通过发展活细胞单分子荧光成像与示踪、超分辨成像等方法，原位表征3-oxo-C12-HSL在细胞膜上的分布，及其对膜上脂质分子分布、TNF-α等受体分子计量比和扩散运动的影响；并利用3-oxo-C12-HSL的系列类似物研究其构效关系。项目的研究结果将对现有的配体介导受体激活理论进行补充与完善，为今后临床上控制和治疗感染以及新药研发提供新的理论与实验依据。

绿脓杆菌是临床上引发术后感染的重要致病菌，它分泌的群体感应信号分子3-oxo-C12-HSL具有抑制宿主免疫细胞的功能，但至今未发现它的结合受体，其作用机制未知。我们发展和应用活细胞单分子荧光成像技术，对TNFR，EGFR及TGFR等受体的单分子扩散运动和聚集状态进行更准确的细胞原位实时表征，发现3-oxo-C12-HSL通过影响膜上受体多聚化激活，来调控宿主应答的分子机制，并证实其作用于不同类型细胞时会影响细胞信号发生和受体內吞，引发下游信号通路的交叉激活，进而产生不同的生物学效应。同时，通过发展体外单层膜的高分辨原子力显微成像技术以及细胞的STED超分辨成像技术，我们观察到3-oxo-C12-HSL具有诱导脂筏结构以及细胞膜张力改变的功能。因此，我们提出并探讨了“非配体依赖的膜诱导受体激活”新原理：即3-oxo-C12-HSL嵌入宿主细胞膜，改变膜上脂筏结构，从而改变膜上受体的单分子行为，影响其信号激活的新观点。我们还利用3-oxo-C12-HSL的系列类似物研究了其构效关系，发现酰基链长和酮基的存在对其生物学功能有重要影响。本项目的研究结果揭示了真核细胞感知微生物代谢产物的一种未知机制，这将对现有的配体介导受体激活理论进行补充与完善，为今后临床上控制和治疗感染，突破多药耐药屏障以及新药研发提供新的理论与实验依据。