σ54蛋白调控幽门螺杆菌适应氧应激的分子机制研究

During the process of infection, transmission and colonization, Helicobacter pylori should be confronted with oxidative stress (oxygen or reactive oxygen species), and the bacterium requires rapid alterations in gene expression to allow itself survival under stress conditions. However, the mechanism underlining this regulation remains unknown. Our study showed that σ54 plays an important role in H. pylori survival of stationary phase. We found that two anti-oxidant proteins Thioredoxin and NapA were positively regulated by σ54, which would benefit H.pylori to defend against oxidative damage in the stationary phase. We also found rpoN null mutant exhibited a marked decrease in viability compared with wild-type H. pylori under oxygen stress. We considered that σ54 protein would play an important regulatory role in H. pylori survival under oxidative stress. On the basis of our previous results, we will conduct deep study on regulatory role of σ54 in H. pylori under oxidative stress as follows: rpoN gene expression at mRNA level and protein level; viability of rpoN null mutant compared with wild-type H. pylori; the killing effect of macrophage on rpoN mutant and wild-type H. pylori.Then microarray will be used to identify the genes regulated by σ54 under oxidative stress. We will use primer extension method to determine that whether the σ54-dependent promoter is exist at upstream of transcription start point of differentially expressed gene. Furthermore, we will utilize ChIP-Seq techniques newly used in bacteria study to acquire the genes directly combined by σ54 at promoter area. Finally, we will study the role of σ54 on bacterial survival in the stomach of mouse in which the oxidative stress environment has been triggered by dead wild-type H. pylori. Our investigations will shed new light on the adaptive regulation of H. pylori under oxidative stress conditions.

作为微需氧的幽门螺杆菌在感染传播过程中经常遭遇氧应激(O2或ROS)，细菌需要快速改变基因表达来适应逆境并存活，然而其适应性调控机制尚不清楚。我们前期研究显示σ54对幽门螺杆菌在稳定期的存活至关重要，σ54通过正向调控两种抗氧化蛋白Thioredoxin和NapA来增强细菌在稳定期的抗氧化能力。并且rpoN（编码σ54）突变株在O2应激下的存活率大大降低，推断σ54对幽门螺杆菌适应体内外遭遇的氧化应激具有重要调节作用，值得深入研究。因此我们拟先证明氧应激下σ54的调节作用，再利用基因芯片技术解析σ54所调控的基因，借助引物延伸法明确差异表达基因上游是否存在σ54依赖的启动子；并结合ChIP-Seq技术锁定σ54能够直接结合在启动子区域并调控其表达的基因。最后对σ54调节幽门螺杆菌在小鼠胃内氧应激环境中存活的作用进行体内研究。本课题将为阐明幽门螺杆菌氧应激条件下适应性生存提供新的分子机制。

作为微需氧的幽门螺杆菌在感染、传播及致病过程中经常遭遇氧应激，幽门螺杆菌的毒力因子等会诱发机体的固有免疫及适应性免疫，使炎症细胞浸润产生大量ROS（活性氧）、NO(活性氮类)。持续的炎症造成感染部位的氧化应激及宿主DNA损伤，长此以往便可能导致慢性胃炎、胃溃疡及胃癌等一些相关疾病的发生。本研究对σ54蛋白是如何调控幽门螺杆菌适应感染微环境中的氧化应激进行了研究。研究发现：与野生株相比，幽门螺杆菌rpoN缺失突变株在正常空气（21%O2）和活性氮（NO）条件下较早出现形态上的衰退，rpoN突变株的存活率大大下降。粘附是细菌在体内定植感染的第一步，结果显示rpoN缺失突变株对AGS上皮细胞的粘附能力与野生株相比大大降低，rpoN缺失突变株诱导的IL-8产生量低于野生株诱导的产生量。以上研究表明σ54蛋白对于幽门螺杆菌在氧应激，粘附定植，以及诱导炎症方面具有重要作用。综合分析转录组测序结果，rpoN缺失突变株中ATP结合蛋白(HP0066)，细菌分裂蛋白(ftsI),以及杆状形态保持蛋白(HP0743)表达显著下降，这可能与突变株存活率下降有关。幽门螺杆菌粘附相关基因HP0009(hopZ)及HP0722(sabB)基因以及鞭毛相关基因在突变株中显著下调，表明σ54蛋白可能通过调控鞭毛基因来调控细菌运动，以及调控HP0009和HP0722基因来调节细菌的粘附。突变株中cag致病岛蛋白表达下降，可能影响毒力因子的释放，再加上粘附的降低，从而使得炎症因子IL-8的产生量降低。该研究表明σ54蛋白可以做为抗菌药物设计的候选靶点。幽门螺杆菌体内定植时也可能遇到一些具有氧化还原性质的物质，如白藜芦醇。白藜芦醇是一种氧化还原活性分子，可与过渡铜离子结合并还原，在此过程中产生活性氧ROS，最终导致DNA损伤。基于此，我们对幽门螺杆菌如何应对白藜芦醇引起的氧化损伤做了相应研究。15个氧化还原相关的基因表达上调，其中包括7个重要的抗氧化蛋白，5个参与维持铁稳态的基因表达上调，一些毒力因子及运动相关基因表达也上调。但未发现σ54蛋白参与白藜芦醇引起的氧化损伤。