线虫病原细菌感知宿主的信号调控机制

It is always the focus field in Biology that research on the the interactions between pathogenic microorganisms and hosts. In our previous study, we found that the pathogenic bacteria Bacillus nematocida B16 had the capabilities to sense the presence of nematodes, regulate their metabolism to form the spores, and finally get ready for trapping nematodes. Although it is a prerequisite for the infection of the recognization of the nematodes by bacteria, the process on the bacteria recognizing the signal molecules produced by nematodes and the signal regulation mechanism has not been reported so far. Thus, here using B16 and C. elegans as research targets, we design to investigate the volatile signal compounds emitted by nematodes using SPME-GC-MS, and then the functions of the compounds were tested individually using commercially available standards. The potential proteins (genes) involved in signaling pathway in the recognization process will be screened out by transcriptome, proteome and bioinformatics analysis, and those proteins involved in singaling regulation of recognization will be further identified by real time PCR (QPCR), gene knockout and complementary experiments. The binding of the signal compounds and the target protein will be investigated by protein purification, ITC and structural biology. The transcriptional regulation of the signal proteins will be explored by EMSA、Footprinting et al. Moreover, the localization of signaling proteins, interactions between different proteins in signaling pathway and the crosstalk among different signaling pathways were confirmed by green fluorescence protein fusion gene technique and the bacterial two-hybrid system. Our research has important scientific significance and application value, since it will help reveal the signaling mechanism regulating the recognition of nematodes in pathogenic bacteria, expand the understanding the interaction mechanism between pathogenic bacteria and their hosts, lay the foundation for rational use of pathogenic bacteria.

病原菌与宿主间的互作关系是生物学研究的前沿和热点。前期发现线虫病原细菌Bacillus nematocida B16可以感知宿主C.elegans的存在，调节相应基因的表达，提前形成芽孢，为诱捕线虫做好准备。虽然细菌感知线虫继而引起两者接触是完成侵染的前提条件，但其中的信号调控机制迄今未见报道。本项目以线虫病原细菌B16和C.elegans为研究对象，采用SPME-GC-MS、纯品验证等鉴定线虫的信号物质；通过转录组、蛋白质组及生物信息学分析筛选B16感知、传递信号物质的蛋白及基因，并通过定量PCR和基因功能实验验证靶基因的功能；采用蛋白纯化、ITC、EMSA、Footprinting、GFP标记和细菌双杂交等技术确定B16关键信号蛋白和信号传导通路；从而揭示病原细菌感知宿主的信号调控机制，拓展人们对病原细菌与宿主的互作机制的认识，为合理利用病原细菌奠定基础，具有重要的科学意义与应用价值。

我们前期在研究杀线虫芽孢杆菌侵染线虫机制过程中发现，芽孢是其完成侵染的重要结构。当以B16的营养细胞侵染线虫时，线虫的死亡率极低，而当以该细菌的芽孢进行生测时，大部分测试线虫快速死亡。本课题聚焦线虫诱导细菌B16产生芽孢的分子机制。（1）线虫诱导的B16在18 h开始形成芽孢，对照组在24 h开始形成芽孢；在32 h时，两者的芽孢形成数目差异最大，实验组的芽孢数目是对照组的66.8倍；（2）SPME-GC-MS检测出6种线虫感知到B16存在时所释放出的信号物质。经过纯品验证试验发现吗啉诱导的B16在18 h开始形成芽孢，对照组在24 h开始形成芽孢，苯酚和十一酸丙酯不诱导B16提前形成芽孢；（3）转录组分析结果表明：实验组32 h和对照组32 h相比，B16 的9个芽孢形成相关基因表达量明显上调，表达量差异倍数在2倍以上。实验组 18 h 和对照组 18 h相比，B16的磷酸酶基因spo0E下调了2 倍以上；实验组32 h 和对照组32 h相比，B16的磷酸酶基因rapA 也下调了2 倍以上。qPCR结果显示，与对照相比，线虫诱导B16 32 h时，spo0E、rapA基因的表达量分别下调58.33倍、47.64倍；吗啉诱导B16 24 h时，spo0E、rapA基因的表达量分别下调9.32倍、2.72倍；（4）分别构建了spo0E、rapA基因在B16中高效表达的菌株，两种基因过表达菌株的产芽孢的能力与野生株相比均有所下降，且用吗啉和线虫诱导过表达菌株，过表达菌株也未能形成芽孢。本研究初步探究了病原细菌B16感知宿主过程中信号调控的关键蛋白和信号通路，为诠释病原细菌开启侵染模式的分子机制奠定理论基础，为进一步从信号调控方面解释细菌感知宿主的机制奠定了良好的基础。该研究内容已经正式发表SCI论文2篇，正式发表中文核心期刊1篇，同时授权国家发明专利2项，还有4项在实审阶段。培养硕士研究生10名。申请人获得河南省学术技术带头人，河南省优秀硕士学位论文指导教师，河南省优秀教师、优秀共产党员称号，并被评为“南阳师范学院卧龙学者”，完成了既定目标。本课题的研究对今后深入探索其他芽孢杆菌感知宿主的差异性机制奠定了良好的基础。