单增李斯特菌plcB缺失株的生物学特性及酵母双杂交诱饵载体构建  

Characterization of Listeria monocytogenes plcB deletion mutant and construction of PlcB-based yeast two-hybrid system

作　　者：黄怡芳 吕洁婷 陈绵绵 程昌勇 宋厚辉 HUANG Yifang;LYU Jieting;CHEN Mianmian;CHENG Changyong;SONG Houhui(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,College of Animal Science and Technology&College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]浙江农林大学动物科技学院/动物医学院浙江省畜禽绿色生态健康养殖应用技术研究重点实验室,浙江杭州311300

出　　处：《中国兽医学报》2021年第7期1328-1334,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31872620,31770040,31972648);浙江省自然科学基金资助项目(LZ19C180001,LQ19C180002);浙江省属高校基本科研业务费专项资金资助项目(2020KJ004)。

摘　　要：为探究单增李斯特菌磷脂酶PlcB在细菌感染过程中的作用及挖掘可能与宿主互作的相关蛋白,本试验利用李斯特菌遗传操作系统构建了plcB缺失回补株及基于PlcB为诱饵的酵母双杂交系统,并通过体外生长曲线、细胞内增殖试验、空斑形成能力等试验研究PlcB介导的生物学功能;通过酵母双杂交诱饵质粒自激活及毒性试验和诱饵蛋白表达检测等方法鉴定可以用于后续互作蛋白筛选的酵母双杂交诱饵质粒。结果显示,缺失plcB基因不影响李斯特菌的体外生长,但缺失株在RAW264.7巨噬细胞内的生存能力相较野生株显著减弱(P<0.01),在L929成纤维细胞中感染形成的空斑较野生株和回补株极显著下降(P<0.001)。将含有pGBKT7-BD-PlcB诱饵质粒的Y2HGold酵母菌株涂布于缺陷培养基中,未发现毒性及自激活现象;通过Western blot能够检测到酵母载体中诱饵蛋白PlcB的稳定表达,说明酵母双杂交系统构建成功,并证实磷脂酶PlcB在李斯特菌宿主内感染和迁移等过程中发挥了重要作用,且基于PlcB的酵母双杂交体系可以为将来深入挖掘PlcB在感染过程中与宿主重要生物学过程及相关分子的互作交流机制奠定了基础。To explore the biological roles and potential host targets of Listeria monocytogenes(L.monocytogenes)phospholipase B(PlcB)during intracellular bacterial infection,the plcB deleted and complemented mutants were characterized,and the PlcB-based yeast two-hybrid bait system was constructed.By using these genetic tools,we investigated the abilities of these mutants in vitro growth,intracellular infection,and cell-to-cell spread in macrophages and fibroblasts.Besides,self-activation and toxicity of the recombinant pGBKT7-BD-PlcB yeast two-hybrid system was tested,and expression of the bait protein PlcB was detected in the yeast cells.The results showed that the deletion of plcB gene did not affect the growth of L.monocytogenes,while the efficiency of intracellular infection in RAW264.7 cells and spread in L929 cells was significantly impaired compared with the wild-type strain.When Y2 HGold yeast strain harboring the pGBKT7-BD-PlcB plasmid was cultured in nutrition-defective media,neither toxicity nor self-activation was found.The stable expression of the recombinant bait PlcB could be detected in the yeast cells by Western blot,confirming that PlcB-based yeast two-hybrid system was successfully constructed.In summary,this study demonstrated the critical role of the phospholipase PlcB in the process of L.monocytogenes infection,more importantly,the PlcB-based yeast hybrid system lays a foundation to further explore the potential host targets as well as the underlying mechanisms exploited by Listeria during the intracellular infection.

关 键 词：单增李斯特菌 磷脂酶B 缺失株 酵母双杂交诱饵载体 胞内感染 细胞间迁移 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]