细菌三型分泌系统转运蛋白的能量利用机制研究

Type III Secretion System(T3SS), a kind of important virulence factors in many bacterial pathogens, is a protein translocating machine which is precise, highly efficient and dynamic. Up to now, most of our knowledge about how to secrete proteins through T3SS comes from the flagellar system, relying on studies using electron-microscopy or biochemical methods. Studies about the dynamic process and mechanism of protein secretion through T3SS are very limited. Here, we propose to use Single Molecule Fluorescence Imaging to monitor how three components of the ATPase complex(MxiN, Spa47 and Spa13) in the Shigella Mxi-Spa system, a typical T3SS, drive protein translocation. We plan to determine the stoichiometry of these three proteins and their exchange rate between the basal body and cytoplasmic pool, and to analyze the free energy utilization from aspects of ATP concentration, proton motive force and pH value. Further, in in vitro and in vivo conditions, we will investigate the molecular basis and energy utilization by triggered T3SS to deliver effector proteins, and compare differences between T3SS and flagella. This program will not only help us learn more about T3SS, but also contribute to developing new drugs targeting T3SS, with vital scientific and medical values.

病原菌的重要毒力因子三型分泌系统是一个精密、高效、动态的蛋白质转运机器。目前研究蛋白分泌机制主要是利用电镜或生化技术在鞭毛系统中完成，而揭示三型分泌系统分泌蛋白的动态过程和机制鲜见报道。本项目将以代表性三型分泌系统--志贺氏菌Mxi–Spa系统为研究对象，采用单分子荧光成像方法，实时、原位、高时空分辨率的观测ATP酶复合物组分(MxiN、Spa47和Spa13)驱动蛋白分泌的动态过程，计算复合物中分子数量与交换频率，从ATP、跨膜电势差和pH值角度全面分析驱动这种过程的能量基础。进一步在志贺氏菌Mxi–Spa系统体内、体外模型中，解析三型分泌系统在激活状态下转运效应蛋白的分子基础和能量利用机制，比较与鞭毛系统在能量利用上的差异。本项目的开展将有助于揭示病原菌中三型分泌系统这类蛋白质机器的能量利用基础，具有重要的科学意义，还有助于开发新的药物作用靶标，具有重要的医学意义。

本项目“细菌三型分泌系统转运蛋白的能量利用机制研究”整体执行顺利，基本完成项目申请时设置的研究目标和研究内容，经费使用符合国家规定。通过本项目的研究，我们发展、优化了适用于单细菌荧光成像的荧光蛋白、荧光染料，在单细菌层面实现细胞膜通透性、跨膜电势差、pH 值、ATP 浓度等不同能量代谢指标的实时测量，来刻画细菌的能量状态。结合高分辨率荧光显微成像技术，以大肠杆菌、溶藻弧菌的三型分泌系统-细菌鞭毛蛋白输出为研究对象，我们实时测量鞭毛蛋白输出速率、细菌鞭毛长度，并建立蛋白质输出与细胞能量状态之间的联系。本研究刻画了典型的细菌三型分泌系统-细菌鞭毛蛋白输出的时空动态过程，揭示了细菌三型分泌系统的能量利用机制。