双组分系统resE/resD基因缺失对单增李斯特菌抗渗透压应激能力及致病性的影响  

Effects of Two-component System resE/resD Gene Deletion on OsmoticStress Resistance and Pathogenicity of Listeria monocytogenes

作　　者：郭骞 张钰 方小伟 袁梅 郑明浩 刘芳 方春 GUO Qian;ZHANG Yu;FANG Xiaowei;YUAN Mei;ZHENG Minghao;LIU Fang;FANG Chun(College of Animal Science,Yangtze University,Jingzhou 434025,China;Animal Health Supervision Institute of Hengshui City,Hengshui 053000,China)

机构地区：[1]长江大学动物科学学院,荆州434025 [2]衡水市动物卫生监督所,衡水053000

出　　处：《中国畜牧兽医》2023年第9期3853-3860,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金青年基金项目(31802208);湖北省教育厅科学技术研究项目(Q20221302)。

摘　　要：【目的】构建单增李斯特菌双组分系统resE/resD基因缺失株,并探究其抗渗透压应激能力及致病性。【方法】利用同源重组方法构建单增李斯特菌双组分系统resE/resD基因缺失株,通过不同环境生长曲线比较分析单增李斯特菌野生株10403S与缺失株ΔresE/resD的生存性能差异;通过应激存活试验探究resE/resD基因缺失对单增李斯特菌抗渗透压能力的影响;通过对结肠癌细胞Caco-2及胃腺癌细胞MGC803进行细胞黏附、侵袭试验以及小鼠毒力试验,评估resE/resD基因缺失对单增李斯特菌细胞黏附与侵袭能力及小鼠致病性的影响。【结果】生长曲线结果显示,resE/resD基因缺失不影响单增李斯特菌正常生长,在5%NaCl条件下缺失株ΔresE/resD生长能力弱于野生株10403S,平台期抗渗透压应激能力显著降低(P<0.05)。应激存活试验结果表明,在10%NaCl条件下应激处理1 h后,缺失株ΔresE/resD存活率极显著低于野生株10403S(P<0.01)。Caco-2及MGC803细胞侵袭试验结果显示,缺失株ΔresE/resD对细胞黏附能力显著减弱(P<0.05),且细胞侵袭能力极显著降低(P<0.01)。小鼠毒力试验结果显示,缺失株ΔresE/resD小鼠脾脏与肝脏中细菌载量极显著或显著低于野生株10403S(P<0.01;P<0.05)。【结论】双组分系统resE/resD基因缺失不影响单增李斯特菌野生株10403S正常生长,但缺失株ΔresE/resD表现出抗渗透压应激能力下降,且减弱了单增李斯特菌对细胞黏附侵袭力和小鼠致病性。【Objective】This study was aimed to investigate the osmotic stress resistance and pathogenicity of Listeria monocytogenes by constructing a two-component system resE/resD gene deletion strain.【Method】The two-component system resE/resD gene deletion strain was constructed by homologous recombination.The growth curve assay of Listeria monocytogenes were analyzed to compare the survival performance of the wild strain 10403S and the deletion strainΔresE/resD.The stress survial test was used to explore the effect of resE/resD gene deletion on the osmotic pressure resistance of Listeria monocytogenes.The effects of resE/resD gene deletion on the cell infiltration ability and pathogenicity of Listeria monocytogenes in mice were evaluated by cell adhesion assay,cell invasion assay and mice virulence assay in colon cancer cells Caco-2 and gastric cancer cells MGC803.【Result】The growth curve assay results showed that resE/resD gene deletion did not affect the normal growth of Listeria monocytogenes.The growth ability ofΔresE/resD in 5%NaCl was lower than that of wild strain 10403S,and the resistance to osmotic stress in platform stage was significantly decreased(P<0.05).The results of the stress survival test showed that the survival rate ofΔresE/resD was extremely significantly lower than that of wild strain 10403S after 1 h of stress treatment in 10%NaCl(P<0.01).In the Caco-2 and MGC803 cell invasion test,the results showed thatΔresE/resD was significantly weaker in cell adhesion(P<0.05),and extremely significantly lower in cell invasion ability(P<0.01).The results of the mice virulence assay showed that the bacterial load of spleen and liver in mice ofΔresE/resD was extremely significantly or significantly lower than that of wild strain 10403S(P<0.01 or P<0.05).【Conclusion】Two-component system resE/resD gene deletion did not affect the normal growth of Listeria monocytogenes wild strain 10403S,butΔresE/resD showed reduced resistance to osmotic stress,and weakened the cell adhesion invasiveness and m

关 键 词：单增李斯特菌 双组分系统 resE/resD基因 渗透压应激 致病力 

分 类 号：S852.617[农业科学—基础兽医学]