单核细胞增生李斯特菌LLO-InlP缺失株的构建及部分生物学特性研究

Construction and biological characterization of an LLO-InlP deletion strain of Listeria monocytogenes

目的构建单核细胞增生李斯特菌LLO-InlP缺失株,探究LLO、InlP基因对单核细胞增生李斯特菌生物学特性的影响。方法通过温度敏感型(Ts)质粒载体基因敲除法,构建单核细胞增生李斯特菌LM681ΔInlP、LM681ΔLLO-InlP基因缺失株,通过检测不同时间的OD 600 nm值绘制生长曲线分析LM681、LM681ΔInlP、LM681ΔInlP-ΔLLO三株菌的体外生长能力;以感染复数MOI值为10感染细胞,测定LM681、LM681ΔInlP、LM681ΔInlP-ΔLLO对人绒毛膜滋养层细胞(HTR-8/SV40)、人绒毛膜癌细胞(JEG-3)黏附侵袭能力;动物水平上测定小鼠半数致死量、感染后的脑、肝、脾载菌量。结果成功获得片段大小为1125 bp的缺失株LM681ΔInlP、LM681ΔInlP-ΔLLO(亲本株LM681为2292 bp);生长曲线结果显示,各菌OD 600值无统计学差异;3株菌体外感染HTR-8/SV40细胞、JEG-3细胞黏附侵袭试验结果显示,LM681ΔInlP、LM681ΔInlP-ΔLLO较LM681对HTR-8/SV40细胞、JEG-3细胞黏附侵袭率均极显著降低(HTR-8/SV40细胞F LM681ΔInlP-LM681=102.792、F_(LM681ΔInlP-ΔLLO-LM681)=128.459,均P<0.01,JEG-3细胞F LM681ΔInlP-LM681=203.755、F_(LM681ΔInlP-ΔLLO-LM681)=81.279,均P<0.01);LM681亲本株的LD_(50)为10^(6.78),LM681ΔInlP缺失株的LD_(50)为107.45,在攻毒剂量范围LM681ΔInlP-ΔLLO不存在LD_(50),LM681ΔInlP-ΔLLO较LM681的LD_(50)提高了6.78个对数数量级,LM681ΔInlP-ΔLLO组织载菌量整体较亲本株LM681降低。结论LLO、InlP基因缺失在动物感染水平和体外培养细胞感染水平上均极大程度的降低了LM的的毒力,且InlP基因对LM681菌株黏附侵袭滋养层细胞具有一定意义,为研究InlP在单核细胞增生李斯特菌致病中的作用奠定基础。

A Listeria monocytogenes LM681ΔLLO-InlP gene deletion strain was constructed through the temperature sensitive(Ts)plasmid vector gene knockout method,and the OD 600 nm values at different times were plotted to analyze the growth curves indicating the in vitro growth ability of three strains of bacteria:LM681,LM681ΔInlP,and LM681ΔInlP-ΔLLO.Cells were infected with each strain at an MOI value of 10.The adhesion and invasion ability were detected in human chorionic trophoblast cells(HTR-8/SV40)and human choriocarcinoma cells(JEG-3).At the animal level,the median lethal dose(LD_(50))in mice,and the bacterial loads in the brain,liver and spleen after infection were measured to explore the effects of the LLO and InlP genes on the biological characteristics of Listeria monocytogenes.The missing strain LM681 with a fragment size of 1125 bpΔInlP,LM681ΔInlP-ΔLLO(parent strain LM681 with 2292 bp)was successfully obtained.The growth curve results indicated no significant difference in OD 600 values among bacteria.The deletion of LLO and InlP genes greatly decreased the virulence of LM in both animal infection and in vitro culture cell infection models.The InlP gene facilitates the LM681 strain’s adherence to,and invasion of,trophoblast cells,thus laying a foundation for studying the role of InlP in the pathogenesis of Listeria monocytogenes.