谷氨酸脱氢酶缺失对单核细胞增生李斯特菌生物被膜、毒力及胞外蛋白表达的影响  

Effect of Glutamate Dehydrogenase Deletion on Biofilm Formation,Virulence and Extracellular Proteins Expression of Listeria monocytogenes

作　　者：王文静[1] 杨丽玉 刘婵娟 赵进 罗勤[1] WANG Wen-jing,YANG Li-yu, LIU Chan-juan, ZHAO Jin, LUO Qin(Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan 430079, China)

机构地区：[1]华中师范大学生命科学学院遗传调控与整合生物学湖北省重点实验室

出　　处：《中国生物工程杂志》2018年第9期1-11,共11页China Biotechnology

基　　金：国家自然科学基金(31571931);遗传调控与整合生物学湖北省重点实验室开放课题(GRIB201709);华中师范大学中央高校基本科研业务费(CCNU18TS024)资助项目

摘　　要：目的：探究谷氨酸脱氢酶缺失对单核细胞增生李斯特菌（Listeria monocytogenes,Lm）生物被膜、毒力及胞外蛋白表达的影响。方法：利用微孔板法,检测谷氨酸脱氢酶缺失对Lm生物被膜形成能力的影响;比较野生株、谷氨酸脱氢酶缺失株EGDeΔgdh A及回复菌株EGDeΔgdh A＋p ERL3-gdh A的溶血活性和对棉铃虫幼虫的半数致死量,检测谷氨酸脱氢酶缺失对Lm毒力的影响;利用i TRAQ技术对Lm野生株EGDe与谷氨酸脱氢酶缺失株EGDeΔgdh A的胞外蛋白进行分离与鉴定,并对差异表达蛋白质进行生物信息学分析。结果：与野生株相比,缺失株形成的生物被膜量显著降低（P≤0.01）,溶血活性下降,对棉铃虫幼虫的半数致死量上升了1.6倍;蛋白质组学结果显示,缺失谷氨酸脱氢酶后差异性表达的胞外蛋白有62个,其中47个蛋白质表达量下调,15个蛋白质表达量上调。进一步将这些差异表达蛋白质同COG数据库进行比对及功能聚类分析,结果显示,这些蛋白质的功能分别属于碳水化合物转运与代谢、核苷酸转运和代谢、能量合成与转运相关、转录及细胞膜细胞壁相关蛋白等16个功能类别;其中碳氮代谢及能量转运相关蛋白数量最多,为24个,占所鉴定出的差异蛋白质总量的38.71%。表明谷氨酸脱氢酶是单核细胞增生李斯特菌中碳氮代谢和能量转运中的关键酶。同时,发现有3个与细菌黏附及生物被膜形成相关的蛋白质表达量下降。以上结果较好地解释了EGDeΔgdh A在基础培养基中生长缓慢和生物量显著性下降,以及生物被膜形成能力降低的现象。结论：谷氨酸脱氢酶在单核细胞增生李斯特菌生物被膜、毒力及胞外蛋白表达方面有重要作用。Objective： To explore the effect of glutamate dehydrogenase deletion on biofilm formation,virulence and extracellular proteins expression of Listeria monocytogenes. Methods： The effect of glutamate dehydrogenase deletion on the formation of L. monocytogenes biofilms was examined using a microtiter plate method. The hemolytic activity,the semi-lethal dose of Helicoverpa armigera（ Hübner） in wild-type,GDHdeficient and revertant strains were compared. The differentially expressed extracellular proteins between the wild strain and GDH-deficient strain were identified and analyzed by i TRAQ technology. Results： Compared with wild strains,the amount of biofilm formed by the GDH-deficient strains was significantly decreased（ P ≤0. 01）,as well as the hemolytic activity,but its semi-lethal dose on Helicoverpa armigera（ Hübner） was increased 1. 6 fold.The functions of all sixty-two differential expressed extracellular proteins identified by i TRAQ were cataloged into16 functional groups, including carbohydrate transport and metabolism, energy synthesis and transport,transcription,cell membrane protein related to cell wall and so on. Among them,the most dominant groups including 24 proteins,accounting for 38. 71% of total differential expressed proteins,are related to carbon and nitrogen metabolism as well as energy transport,suggesting that glutamate dehydrogenase is a key enzyme in carbon and nitrogen metabolism as well as energy synthesis and transport for L. monocytogenes. The slow growth and reduced biomass of EGDeΔgdh A compared to EGDe in minimal essential medium were in agreement well with these proteomic results. In addition,the expressions of three proteins from EGDeΔgdh A associated with bacterial adhesion and biofilm formation were also significantly decreased. Conclusion： Deletion of glutamate dehydrogenase reduces bacterial biofilm formation and virulence,affects significantly the extracellular protein expression in L.monocytogenes.

关 键 词：单核细胞增生李斯特菌 谷氨酸脱氢酶 生物被膜 毒力 ITRAQ 胞外蛋白表达 

分 类 号：Q819[生物学—生物工程]