致羔羊脑膜炎型大肠杆菌ompA基因缺失菌株的构建及其致病性

Construction of encephalitis-pathogenic NMGCF-19 E.coli strain with ompA deleted and its pathogenicity

应用CRISPR/Cas9技术构建NMGCF-19ΔompA菌株,并应用小鼠模型确定ompA基因在NMGCF-19菌株感染小鼠中的作用及其机制。结果显示,与野生型菌株比较,ompA基因敲除菌株感染小鼠的海马体神经元细胞坏死明显降低,且不呈局灶性;同时,脑组织中菌体检出量明显减少,紧密连接蛋白ZO-1和Occludin的mRNA水平和蛋白表达水平均明显提高。结果表明,ompA基因敲除后NMGCF-19菌株其致病性降低,该毒力基因ompA在NMGCF-19侵袭小鼠血脑屏障的过程中发挥重要作用。

The NMGCF-19 strain is an E.coli strain isolated and identified in our laboratory from lambs manifesting severe diarrhea and meningitis.Previous analysis of the genome sequence of NMGCF-19 strain showed that the outer membrane protein A(ompA)gene was a potential virulent gene.In order to determine whether the ompA gene is associated with the pathogenicity of NMGCF-19 strain and the underlying mechanism,the NMGCF-19 strain with ompA knockout(NMGCF-19ΔompA)was generated in this study using CRISPR/Cas9 technology and used to determine the role of ompA gene in mediating the encephalitis by NMGCF-19 infection and the underlying mechanism using the mouse model system.The results showed that the neuronal cell necrosis in the hippocampus in mice infected by NMGCF-19ΔompA was significantly reduced and was not focal compared with that of mice infected with the wild-type NMGCF-19 strain.The number of bacteria in brain of mice infected by NMGCF-19ΔompA was significantly reduced in comparison to that of mice infected by NMGCF-19.Simultaneously,the mRNA and protein expression levels of the tight junction proteins ZO-1 and Occludin were both increased in mice infected by NMGCF-19ΔompA strain compared with the mice infected by NMGCF-19 strain.These results suggest that the ompA gene is a virulent gene and plays an important role in the invasion of the blood-brain barrier by NMGCF-19 strain in mice.