全局调控因子FNR对产肠毒素大肠杆菌生物学特性影响的研究  

Effects of global regulatory factor FNR on biological characteristics of Enterotoxigenic Escherichia coli

作　　者：杨延 毛曌 江丰伟 李干武 YANG Yan;MAO Zhao;JIANG Feng-wei;LI Gan-wu(Veterinary Public Health Key Laboratory of the Ministry of Agricultural,State Key Laboratory of Veterinary Biotechnology,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第9期977-983,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(32002257)。

摘　　要：为研究全局调控因子富马酸盐和硝酸盐减少调控蛋白(FNR)对产肠毒素大肠杆菌(ETEC)生物学特性的影响,本研究通过Red同源重组方法构建了ETEC MD724-020菌株fnr基因的缺失株Δfnr及回补株Δfnr(pfnr),分别将ETEC野生株(WT)、Δfnr以及Δfnr(pfnr)同时接种于TSB液体培养基培养,测定fnr基因缺失前后ETEC的生长曲线,结果显示,与野生株相比,缺失fnr基因后其生长速率无明显变化。将3株菌分别接种于TSB固体培养基,通过测定游走直径比较分析野生株、缺失株和回补株的运动性,结果显示,与野生株相比,缺失株Δfnr的运动能力显著降低(P<0.05)。采用结晶紫法检测3株菌生物被膜(BF)形成能力,结果显示,相比WT和Δfnr(pfnr)株,缺失株Δfnr BF形成能力极显著下降(P<0.001)。将3株菌分别于含10%绵羊血的TSA平板上划线培养,进行溶血性试验,结果显示,相比WT和Δfnr(pfnr)株,Δfnr株溶血圈较野生株显著缩小。将3株菌分别与猪小肠上皮细胞IPEC-J2互作2 h后,通过平板计数法分析各菌株对该细胞的黏附能力,结果显示,与野生株相比,缺失株Δfnr对IPEC-J2细胞的黏附能力显著减弱(P<0.05)。提取上述互作后未黏附细菌的RNA,通过荧光定量PCR检测fnr基因缺失后对致病性ETEC相关毒力基因转录水平的影响,结果显示,与野生株相比,Δfnr株中eaeH、eltA和hlyA基因的转录水平均极显著下降(P<0.001)。本研究首次对全局调控因子FNR影响ETEC的运动性、溶血性、BF形成能力等生物学特性进行了研究,为进一步开展ETEC致病机理的研究提供了参考依据。In order to study the effects of FNR on biological characteristics of ETEC MD724-020 strain,the fnr mutantΔfnr and its complemented strainΔfnr(pfnr)were constructed by Red homologous recombination method.ETEC wild strains(WT),Δfnr andΔfnr(pfnr)were inculated in TSB liquid medium to detect the growth curves of ETEC before and after FNR gene deletion.The results showed that compared with the wild strain,the growth rate of ETEC with FNR gene deletion did not change significantly.Subsequently,three strains were inoculated in TSB solid medium,respectively,and the motility and biofilm formation(BF)ability of the wild type(WT),Δfnr andΔfnr(pfnr)strains were compared and analyzed.The results showed that compared with the wild strain,the motility ability of theΔfnr was reduced(P<0.05),and its BF formation ability was also significantly reduced(P<0.001);Three strains were cultured on TSA plates containing 10%sheep blood,and the resulsts showed that theΔfnr hemolytic circle was significantly smaller than that of the wild strain.After three strains were interacted with porcine small intestinal epithelial cells IPEC-J2 for 2 hours,the adhesion ability of each strain to IPEC-J2 cells was anlysed by plate counting method.The results showed that the adhesion ability ofΔfnr to IPEC-J2 cells was decreased(P<0.05).Compared with the wild-type strain,the transcription levels of eaeH,eltA and hlyA inΔfnr strain was significantly decreased(P<0.001)by quantitative real-time PCR.These results show that the global regulation FNR affects the biological characteristics of ETEC such as motility,hemolysis,and BF formation ability,which provides a reference for further study of the function of FNR as well as pathogenic mechanism of ETEC.

关 键 词：产肠毒素大肠杆菌 FNR 生物学特性 

分 类 号：S852.61[农业科学—基础兽医学]