幽门螺旋杆菌鞭毛马达开关蛋白的化学计量分析

All gastric Helicobacter species possess a prominent feature of flagella-driven motility that is essential for colonization and infection in those tested. Flagellar assembly, export, torque generation and chemotactic induced-switching are controlled by the flagellar motor switch complex typically composed of FliG, FliM and FliN. While the core components of the motor are generally homologous, there is growing genomics, transcriptomic and biophysical evidence suggesting structural diversity of bacterial flagellar motors. Interestingly and unusually, H. pylori harbors an additional switch protein FliY and a unique set of chemotactic components that may associate with its ability to colonize and move in the acidic and viscous gastric mucus layer. Although the crystal structures of various switch proteins from thermophilic bacteria have been resolved, how they assembled and coordinated to change the rotation direction is unclear. In particular, our knowledge of the flagellar system in H. pylori is limited. We are particularly interested in understanding the molecular architecture of flagellar motor in this microbe. Recently, we have solved the crystal structures of chemotactic protein CheY, motor switch protein FliG, FliM and FliG-FliM complex from H. pylori. We have further demonstrated the molecular interaction of CheY-FliM, FliG-FliM and FliY-FliN, and mapped the domains required for their interactions. Using in vivo molecular genetic studies, we have also characterized the biological importance of FliY in flagella formation and function. As an extension to our existing findings, in this study we will apply a combination of molecular microbiology and biophysics approaches to investigate the stoichiometry of motor switch proteins and characterize the dynamic nature of the motor upon chemotaxis in H. pylori. Specifically, we will construct complementation strains with respective switch proteins fused with a fluorescent protein tag. Quantitative total internal reflection fluorescent microscopy will be adopted to analyze the subunit stoichiometry of each switch protein in cells. Furthermore, population of FliM in the "static" and "dynamic" states will also be studied upon addition of chemotactic attractant. Our work will generate novel knowledge, for the first time, about the molecular structure and dynamic behavior of flagellar motor in H. pylori. It will also lay a foundation to characterize the roles of chemotactic components in this pathogenic microbe and to understand the diversity of the bacterial flagellar motor systems.

肠胃性螺杆菌属细菌借助鞭毛提供动力以促进细菌定植及侵染宿主细胞。鞭毛分子马达由不同开关蛋白组成，可控制鞭毛组装、动力输出及趋性反应。然而目前对于细菌鞭毛系统的了解十分局限。值得一提的是，幽门螺旋杆菌具有特有的开关蛋白FliY和特殊的趋化组件。为了研究幽门螺旋杆菌的鞭毛马达系统，我们将结合分子微生物学和生物物理学方法，对分子马达中的开关蛋白进行化学计量分析，以揭示幽门螺旋杆菌分子马达的静态及动态特征。具体地说，我们将利用现有的各开关蛋白的空突变株构建含有荧光蛋白标签的相应互补株，利用全内反射荧光显微镜对细胞中的开关蛋白进行定量分析。再者，将使用趋化引诱剂，以研究FliM与分子马达结合及脱离的动态变化。本课题将首次从新的角度阐述幽门螺旋杆菌鞭毛马达系统的分子结构及动态特征，同时研究细菌趋化性与鞭毛马达系统之间的相互关系。本课题的研究将为日后细菌鞭毛马达系统的多样性研究提供重要实验基础。

所有经过测试的胃螺杆菌都具有鞭毛驱动的运动性。这个显着特征对于它们的定植和感染至关重要。鞭毛组装、蛋白输出、动力产生和趋化诱导的旋转方向转换通常是由FliG、FliM和FliN组成的鞭毛马达开关复合体控制。虽然马达的核心部件通常是同源的，但是越来越多的基因组、转录组学和生物物理学证据表明细菌鞭毛马达具有结构多样性。有意思并且不同寻常的是，幽门螺旋杆菌含有额外的开关蛋白FliY和一组独特的趋化性成分，这可能与其在酸性且粘稠的胃粘液层中的定植和运动性有关。尽管嗜热细菌的各种开关蛋白的晶体结构已经被解析，但它们如何组装和协调以改变其旋转方向的机制还不清楚。特别的是，我们对幽门螺杆菌的鞭毛系统的了解非常有限。因此，我们对其鞭毛马达的分子结构特别感兴趣。..在这项研究中，我们综合运用分子微生物学和生物物理学方法来研究幽门螺杆菌中马达转换蛋白的相互作用和化学计量。具体而言，我们已经分离了一种拟亚精胺合成酶SpeE作为FliM的相互作用蛋白。 SpeE敲除菌株偏好顺时针旋转方向，表明SpeE是马达运动方向转换的的调制蛋白。此外，我们还通过共纯化证明FliY-FliN和FliY-FliM可形成异源二聚体。光散射和分析超速离心的研究进一步证实它们以1：1的化学计量相互作用。我们通过晶体学研究揭示了FliM-SpeE和FliY-FliN复合物的原子结构。此研究结果加深了我们对幽门螺旋杆菌中马达组装和调节的认识。物种特异性的大分子组装暗示在不同环境因素下鞭毛发育的不同遗传调控，这在将来会有更深入的研究。