eha基因调控迟缓爱德华菌抵抗巨噬细胞氧化杀菌作用

ehagene is required for Edwardsiella tarda oxidative stress resistance in macrophage

目的迟缓爱德华菌(E.tarda)能够在巨噬细胞内生存和繁殖,必须抵抗细胞产生活性氧的杀菌作用。eha基因是该菌一个重要的转录调控基因,本研究探讨eha基因调控E.tarda抵抗巨噬细胞氧化杀菌的机制。方法光镜观察野生株和缺失株分别感染RAW264.7巨噬细胞的过程,及菌落计数法测定细菌胞内存活数目;测定细菌在不同H2O2浓度中的存活率;流式法检测细菌感染巨噬细胞后产生活性氧的细胞比率;qRT-PCR测定细菌超氧化物歧化酶基因sodC和过氧化氢酶基因katB的转录水平。结果 eha基因的缺失,使E.tarda在巨噬细胞内的繁殖速率明显下降(P<0.05),使该菌在不同H2O2浓度中的存活率显著降低(P<0.05),使该菌感染细胞组产生活性氧的细胞比率升高;qRT-PCR结果显示,eha基因的缺失使得该菌的超氧化物歧化酶基因sodC和过氧化氢酶基因katB转录水平下降。结论 eha基因通过调控E.tarda菌分解H2O2相关基因的表达,从而影响该菌分解巨噬细胞中活性氧的能力和在胞内外的存活率。因此,eha基因调控了E.tarda菌抵抗巨噬细胞内氧化杀菌作用,有助于该菌在巨噬细胞内生存和繁殖。

As Edwardsiella tarda（E.tarda）can survive and replicate in macrophages,it must resist against reactive oxygen species（ROS）produced by the cells.The haemolysin activator gene（eha）is an important transcriptive regulating gene.Our researches studied the mechanism for the ehagene how to regulate E.tardato resist oxidation stress in macrophages.After the bacteria have infected RAW264.7macrophages,bacterial colonies were counted on LB plates.The results of the above experiment and the observation under optical microscope indicated that both the wild and theΔehastrains can survive and multiply within macrophages,and the reproductive rate of theΔeha within macrophages decreased obviously than that of the wild.The survival rate of theΔehain H2O2 was less than that of the wild.After the level of ROS from macrophages infected with bacteria were detected by flow cytometry（FCM）,the ROS from the macrophages infected with theΔeha（65.49%）was more than that of the wild（55.32%）.The qRT-PCR results showed further that the mutation of the eha gene regulated down the expressions of catalase and superoxide dismutase genes in E.tarda.Therefore,the results indicated that ehagene has affected bacterial ability of decomposing ROS in macrophages and survival ability in H2O2,as the gene may control the expressions of catalase and superoxide dismutase genes in E.tarda.