转录调控因子Fur直接调控鸭疫里氏杆菌毒力的研究

The Riemerella anatipestifer infection is one of the most important diseases to duck industry in our country, which usual occurs as an acute, contagious, septic disease in ducks. Ferric-uptake regulator protein (Fur) was an important global regulator for pathogenic bacteria, which could regulate various vital movements including growth, metabolism and virulence and so on. However, which kind of regulatory mechanism of Fur in virulence of R. anatipestifer is still unclear. In present study, we will investigate the directly regulatory function of the Fur protein in virulence of R. anatipestifer. And the following experiments will conduct: Construction of an isogenic knockout mutant of fur gene in RA-YM using the homologous recombination protocol, and evaluation of the impact of fur deletion on the biological characteristics and virulence. Using deep sequencing technology and quantitative PCR, the transcription profiles of the RA-YM under iron starvation condition with those supplemented with excessive iron, the RA-YM with the fur deficient mutant under iron rich condition and in vivo are compared to define genes controlled by Fur. The subsequent bioinformatical analysis and biochemical experiments enable the systemic characterization of Fur modulon. Taking all the above results together, we will reveal the directly regulatory mode of Fur in the virulence of R. anatipestifer. The result will facilitate the understanding of gene function which regulated by Fur and provides experimental evidence for investigation of their virulence synergy mechanism.

鸭疫里氏杆菌(Riemerella anantipestifer，RA)病是由鸭疫里氏杆菌引起的雏鸭的一种急性、接触性败血性传染病，是对我国水禽业危害最为严重的传染病之一。铁摄取调节蛋白Fur是细菌重要的全局转录调控因子，调节细菌的生长、代谢和毒力等多种生命活动。目前对Fur是通过怎样的机制来调控RA的毒力尚不清楚。因此，本研究拟开展转录调控因子Fur直接调控RA毒力的研究，通过基因缺失构建血清1型鸭疫里氏杆菌RA-YM fur突变株，分析Fur缺失对RA-YM表型和毒力的影响；采用RNA-seq技术比较RA-YM在铁缺乏与铁富余条件下、RA-YM与fur突变株在铁富余条件和体内感染条件下的全局转录谱差异，筛选出Fur调控的基因；通过生物信息学和生化分析，确定Fur直接调控的基因，揭示Fur直接调控RA毒力的模式，为进一步明确Fur调控基因的功能及其在RA毒力中协同作用的机制提供实验证据。

鸭疫里氏杆菌是危害我国养鸭业的重要细菌性病原，目前对其致病机理还不清楚。铁摄取调节蛋白Fur参与调控细菌的生长、代谢和毒力等多种生命活动。本研究开展了Fur直接调控鸭疫里氏杆菌毒力的研究，取得的结果如下：.1. 建立了以苯丙氨酸-tRNA合成酶pheS为负向筛选标记的鸭疫里氏杆菌无抗性基因缺失方法，构建了fur基因缺失突变株△fur。△fur与亲本株RA-YM的生长特性和生化特性没有差异；与亲本株相比，△fur的毒力下降了80倍，其互补菌株△fur（pCP29-fur）毒力恢复；△fur对组织的侵袭力和病变比亲本株下降，表明Fur为鸭疫里氏杆菌的毒力因子。.2. 利用RNA-seq技术系统获得鸭疫里氏杆菌68个铁调控基因和11个Fur直接调控的基因。铁调控和Fur直接调控基因的编码产物涉及氨基酸与辅基生物合成、细胞表面结构与修饰、合成与代谢、转录调控、转运和结合蛋白和未知功能等。在低铁环境中，鸭疫里氏杆菌PorSS分泌系统组件SprT基因表达量上调，提示铁参与鸭疫里氏杆菌蛋白分泌的调控。对12个基因的表达水平进行了qRT-PCR验证，与转录组测序结果一致。预测Fur结合位点序列为ATTTAGAATTATTCTAAAT。通过凝胶阻滞实验明确了hmuR、RAYM_01847、RAYM_00450和RAYM_09824等4个基因直接受Fur调控。.3. 构建了铁调控基因RAYM_00365基因缺失突变株。ΔRAYM_00365与亲本株在对数生长期的生长速度差异不明显，生化特性没有发生改变；突变株在含H2O2、胆盐和酸性环境下生长变慢，表明其对环境胁迫（H2O2、胆盐、酸性）的耐受能力降低；突变株毒力与亲本株没有差异。.4. 构建了铁调控基因sprT基因缺失突变株ΔsprT。ΔsprT的生长特性、生化特性和高盐耐受能力和亲本株一致；ΔsprT的血清存活率显著高于亲本株。与亲本株相比，ΔsprT 毒力下降了4.21×104倍；ΔsprT的组织载菌量及病变程度显著比亲本株轻微；ΔsprT能够对亲本株10LD50攻毒产生良好保护。明胶酶谱试验显示ΔsprT不能够分泌明胶酶。SprT是鸭疫里氏杆菌PorSS分泌系统关键组件，其缺失突变后致毒力显著下降，表明通过PorSS系统分泌的蛋白是鸭疫里氏杆菌重要的毒力因子。.在本项目的资助下在PLOS ONE上提交论文一篇，培养研究生3名。