黄单胞菌组氨酸激酶VgrS感应金属离子的特异性及在植物病害发生中的作用机制

Metal is essential to the survival and infection of all bacterial pathogens, phytopathogenic bacteria. Metal replete and depleted environments are harmful to bacteria, and thus they have to effectively sense the concentrations of different metals and react adaptively. However, the biochemical process of metal recognition remains an opening question. In the previous study, we have found that the receptor histidine kinase, VgrS, which constitutes the VgrS-VgrR two-component signal tranduction system of Xanthomonas campestris pv. campestris, the causative agent of black rot disease of crucifers, is a extracytoplasmic receptor to directly detect not only Fe, but also Cu. In addition, the autokinase activity of VgrS is also affected by other metals, such as Mn, Zn, and Co, albeit these metals did not bind to the signal input region of VgrS. To explore the roles of VgrS-metal interaction in bacterial infection, the current project will combine genetic, enzymological, and biophysical approaches to investigate the recognition spectrum of metals, identify the binding site in VgrS, and dynamics of protein-metal (especially iron and copper) interactions. These results will facilitate the in-depth research on the adaptive responses of the bacterium when facing to the oxidative stress and hypoxia stress in the host plant. In addition, we will also compare the capability of metal recognition of VgrS orthologues between different species of Xanthomonas spp. The results from this project will give insight into how a phytopathogenic bacterium recognize its metal world, and will promote the development of novel anti-bacterial, metal-containing chemicals to defend these important plant pathogens.

金属离子是病原细菌在植物体内生存和感染所必需的元素。金属离子匮乏或浓度过高均会导致细菌死亡。然而，细菌如何特异性地识别金属离子并调控体内离子平衡并不清楚。前期研究发现野油菜黄单胞菌的双组分信号转导系统vgrS-vgrR不但能识别铁，而且也能识别铜和感应其它金属离子，并对细菌毒力进行调控。为了深入分析受体组氨酸激酶VgrS识别多种金属离子的分子机制及在致病调控中的功能，拟通过遗传学、酶学和生物物理学方法，系统分析VgrS识别不同金属离子（特别是铁和铜）的能力、特异性结合位点、受体－离子间相互作用的动力学特征及调控下游基因转录的分子机制。在此基础上，进一步分析VgrS-离子相互作用在寄主感染过程中适应低氧胁迫和氧化胁迫的调控功能，分析病原细菌物种分化过程中金属离子识别谱变异的生物学意义。项目结果将为理解植物病原细菌识别多种金属离子的生物化学机制，以及发展新型抑菌金属制剂提供分子证据。

先前的研究表明野油菜黄单胞菌（Xcc 8004）中的双组分系统VgrSR能够直接感知宿主环境的铁缺乏条件，抑制下游铁转出系统基因tdvA的表达，维持病原细胞的铁稳态。本项目首先通过丙氨酸扫描突变结合生物化学和生物信息学分析，证明VgrS感应区的9个位点对Fe结合和VgrS酶活性至关重要，其中7个单点突变降低了铁结合亲和力和缺铁诱导的蛋白质磷酸化水平。此外，我们还证明了VgrS同样能够直接感知环境中Zn离子的浓度变化。与Fe相比，VgrS感应区对Zn敏感的重要位点有4个，他们影响了Zn匮乏诱导的蛋白构象变化和酶活性，以此维持体内Zn的平衡。VgrSR调控途径研究表明，VgrSR还能协同铁结合抑制子Fur在缺Fe时激活细菌T3SS分泌系统基因和效应子的表达，富铁时抑制效应子的表达。进一步项目筛选了靶向VgrS的植物源杀菌剂。通过以上研究，本项目完成了立项书提出的科学问题，即VgrS感应不同金属离子的特异性及与致病相关基因的调控关系，多种金属离子协同调节VgrS受体激酶的分子机制。项目揭示了双组分系统响应寄主植物金属离子调节细菌致病性、刺激植物免疫的具体方式，为靶向细菌双组分系统的新型临床杀菌和农业防治细菌病害提供重要的分子生物学证据。