单核细胞增生性李斯特氏菌tatD基因缺失对小鼠毒力和肠道菌群的影响  

Effect of tatD gene deletion of Listeria monocytogenes on virulence and gut microflora in mice

作　　者：牛俊辉 李琦 毛福超 钱满 贾艳艳[1,2,3] 丁轲[1,2,3] 张春杰[1,2,3] 程相朝[1,2,3] 廖成水[1,2,3] NIU Junhui;LI Qi;MAO Fuchao;QIAN Man;JIA Yanyan;DING Ke;ZHANG Chunjie;CHENG Xiangzhao;LIAO Chengshui(Laboratory of Functional Microbiology and Animal Health,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,Henan,China;Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control,Henan University of Science and Technology,Luoyang 471023,Henan,China;The Key Lab of Animal Disease and Public Health,Henan University of Science and Technology,Luoyang 471023,Henan,China)

机构地区：[1]河南科技大学动物科技学院功能微生物与畜禽健康实验室,河南洛阳471023 [2]河南科技大学洛阳市活载体生物材料与动物疫病防控重点实验室,河南洛阳471023 [3]河南科技大学动物疫病与公共卫生重点实验室,河南洛阳471023

出　　处：《浙江农林大学学报》2024年第1期161-168,共8页Journal of Zhejiang A&F University

基　　金：河南省科技攻关项目(182102110061);河南科技大学研究生创新基金项目(CXJJ-2021-KJ07);河南科技大学青年骨干教师培养计划(13450009)。

摘　　要：【目的】探究单核细胞增生性李斯特氏菌Listeria monocytogenes tatD基因缺失对小鼠Mus musculus毒力和肠道菌群的影响,为tatD在单核细胞增生性李斯特氏菌与宿主互作中的作用及减毒疫苗研究提供参考。【方法】采用Bliss法将亲本菌株(LM10403s)、缺失菌株(LM10403sΔtatD)和互补菌株(LM10403sCΔtatD)口服感染小鼠,确定菌株对小鼠的半数致死量(LD_(50))。LM10403sΔtatD以1.00×10^(5) CFU口服免疫小鼠观察其免疫保护效果。将40只6周龄雌鼠随机平均分为对照即磷酸缓冲盐溶液组(PBS)、LM10403s组、LM10403sΔtatD组和LM10403sCΔtatD组,PBS组小鼠灌胃200μL无菌PBS,实验组分别灌胃200μL含1.00×10^(6) CFU的菌液。灌胃24 h后剖杀各组小鼠,收集肠道内容物,采用Illumina Hiseq测序技术测定各处理小鼠盲肠样品微生物16S rRNA V3-V4区序列,并比较分析其微生物群落结构、多样性和信号通路富集。【结果】LM10403sΔtatD的LD_(50)为8.11×10^(7) CFU,毒力低于LM10403s。同时LM10403sΔtatD口服免疫小鼠后对单核细胞增生性李斯特氏菌亲本菌株的攻毒能提供80%保护率。Chao1和Observed species指数显示:PBS与LM10403sΔtatD处理组差异不显著,但LM10403s组和LM10403sCΔtatD处理组相比于PBS处理组显著下降(P<0.05)。在门水平上,LM10403sΔtatD处理组的厚壁菌门Firmicutes相对丰度高于LM10403s和LM10403sCΔtatD处理组,而在属水平上,乳杆菌属Lactobacillus相对丰度高于LM10403s和LM10403sCΔtatD处理组。功能预测分析显示:LM10403s处理组在细胞过程、环境信息处理和新陈代谢等信号通路的富集程度高于LM10403sΔtatD处理组。【结论】tatD基因缺失后单核细胞增生性李斯特氏菌的毒力降低,并具有一定的免疫保护效果。tatD基因缺失菌株感染小鼠对肠道菌群失调影响减小,且有益菌增多。[Objective]This study,with an investigation of the effects of tatD gene deletion in Listeria monocytogenes on virulence and gut microbiota in mice(Mus musculus),is aimed to provide reference for the role of tatD in the interaction between L.monocytogenes and the host,as well as for the study of attenuated vaccines.[Method]First,mice were orally infected with LM10403s,LM10403sΔtatD and LM10403sCΔtatD strains using the Bliss method to determine the strain’s median lethal dose(LD_(50))for mice before immunoprotective effect of LM10403sΔtatD was observed in mice immunized with 1.00×10^(5) CFU orally.Then,forty 6-week-old female mice were randomly and equally divided into PBS(ck),LM10403s,LM10403sΔtatD,and LM10403sCΔtatD groups,with those in the ck group gavaged with 200μL of sterile PBS and the ones in the experimental group gavaged with 200μL of bacterial solution containing 1.00×10^(6) CFU,respectively.Next,after 24 h of gavage,the mice in each group were dissected and killed,with intestinal contents collected before Illumina Hiseq sequencing technology was used to determine the sequences of the microbial 16S rRNA V3−V4 regions of the cecum samples from each group so that a comparative analysis was conducted of the structure of microbial communities,diversity,and signaling pathway enrichment.[Result]The LD_(50) of LM10403sΔtatD was 8.11×10^(7) CFU,which was less virulent than that of the parental strain.Oral immunization of mice with LM10403sΔtatD provided 80%protection against L.monocytogenes parental strain infection.Chao1 and Observed species indices showed that the difference between ck and LM10403sΔtatD group was not significant,but LM10403s and LM10403sCΔtatD group were significantly lower compared to ck group(P<0.05).At the phylum level,the relative abundance of the Firmicutes in LM10403sΔtatD group was higher than that in LM10403s and LM10403sCΔtatD group,and at the genus level,the relative abundance of the genus,Lactobacillus in LM10403sΔtatD group was higher than that in LM10403s and

关 键 词：tatD基因 单核细胞增生性李斯特氏菌 毒力 16S rRNA 肠道菌群 

分 类 号：S811.6[农业科学—畜牧学]