单增李斯特菌溶血素S llsB基因缺失株的构建及部分生物学特性研究

doi:10.16431/j.cnki.1671-7236.2018.12.006

摘要/Abstract

摘要：

为分析单增李斯特菌溶血素S （listeriolysin S，LLS）llsB基因缺失对单增李斯特菌（Listeria monocytogenes，LM）生物学特性的影响，本研究利用同源重组构建了llsB基因缺失株LM90-ΔllsB，并以8周龄、体重40 g±5 g的健康昆明系雄性小鼠为试验动物对其生长特性、半数致死量（LD₅₀）、脏器载菌量等生物学特性进行了初步研究。结果显示，本研究成功构建了llsB基因缺失株；缺失株在体外连续培养20代过程中能稳定遗传。通过生长曲线测定发现，llsB基因缺失株生长活性略高于亲本株；小鼠感染试验结果显示，亲本株和缺失株的LD₅₀分别为10^6.17和10^6.50 CFU；与亲本株相比，缺失株在小鼠肝脏和脾脏中的载菌量均极显著减少（P<0.01），说明缺失株对小鼠的感染能力明显减弱。在小鼠脑中未检出单增李斯特菌。综上所述，llsB基因对LM90的部分生物学特性可能有直接或间接的调控作用，本试验结果为进一步研究LLS的致病机理及防控李斯特菌病提供一定的理论依据。

关键词: 单增李斯特菌溶血素S (LLS); llsB基因; 基因缺失株; 生物学特性

Abstract:

In order to analyze the effect of listeriolysin S (LLS) llsB gene deletion on the biological characteristics of Listeria monocytogenes (LM),this study used homologous recombination to construct the llsB gene deletion strain LM90-ΔllsB,and the biological characteristics of growth characteristics,median lethal dose (LD₅₀) and organ-borne bacteria were studied in healthy Kunming male mice at 8 weeks old and weighing 40 g±5 g.The llsB gene deletion strain was successfully constructed,and the deletion strain had good genetic stability through continuous passage to 20 generations in vitro.Based on its growth curve examination,we found that the growth rate of the mutant strain was slightly higher than that of the parent strain.The results of mice infection test showed that the LD₅₀ of the parent strain and the deletion strain were 10^6.17 and 10^6.50 CFU,respectively.Compared with the parent strain,the amount of bacteria load of the deletion strain in the liver and spleen of the mice was extremely significantly decreased (P<0.01),the results showed that the infection ability of the mutant strain on mice was obviously weakened.No Listeria monocytogenes was detected in the brain.The results suggested that llsB gene might have direct or indirect regulatory effect on some biological characteristics of LM90,and it would provide a theoretical basis for further understanding of the pathogenic mechanism of LLS,prevention and control of listeriosis.

Key words: listeriolysin S (LLS); llsB gene; gene mutant; biological characteristics

中图分类号:

S852.61

尹华, 任静静, 王欣雨, 蒋建军. 单增李斯特菌溶血素S llsB基因缺失株的构建及部分生物学特性研究[J]. 《中国畜牧兽医》, 2018, 45(12): 3355-3362.

YIN Hua, REN Jingjing, WANG Xinyu, JIANG Jianjun. Construction and Partial Biological Characteristics Analysis of Listeriolysin S llsB Gene Deletion Strain[J]. , 2018, 45(12): 3355-3362.

参考文献

[1] JEMMI T,STEPHAN R.Listeria monocytogenes:Food-borne pathogen and hygiene indicator[J].Revue Scientifique et Technique,2006,25(2):571-580.
[2] 杜冬冬,张奇文,李红欢,等.单增李斯特菌lmo0331基因的克隆、序列分析及原核表达[J].中国畜牧兽医,2018,45(5):1170-1176. DU D D,ZHANG Q W,LI H H,et al.Cloning,sequence analysis and prokaryotic expression of lmo0331 gene from Listeria monocytogenes[J].China Animal Husbandry & Veterinary Medicine, 2018,45(5):1170-1176.(in Chinese)
[3] 江玲丽,陈健舜,方维焕,等.单核细胞增多性李斯特菌主要毒力因子研究进展[J].中国人兽共患病学报,2003,23(7):733-735. JIANG L L,CHEN J S,FANG W H.Advances in the study of main virulence factors of Listeria monocytogenes[J].Chinese Journal of Zoonosis,2007,23(7):733-735.(in Chinese)
[4] 陈健舜,江玲丽,方维焕.李斯特菌毒力因子及其进化[J].微生物学报,2007,47(4):738-742. CHEN J S,JIANG L L,FANG W H.Virulence factor and its evolution of Listeria sp.[J].Journal of Microbiology,2007,47(4):738-742.(in Chinese)
[5] VOGEL B F,HUSS H H,OJENIYI B,et al.Elucidation of Listeria monocytogenes contamination routes in cold-smoked salmon processing plants detected,by DNA-based typing methods[J].Applied & Environmental Microbiology,2001,67(6):2586-2595.
[6] VLAEMYNCK G,LAFARGE V,SCOTTER S.Improvement of the detection of Listeria monocytogenes by the application of ALOA,a diagnostic,chromogenic isolation medium[J].Journal of Applied Microbio-logy,2000,88(3):430-441.
[7] VÁZQUEZBOLAND J A,KUHN M,BERCHE P,et al.Listeria pathogenesis and molecular virulence determinants[J].Clinical Microbiology Reviews,2001,14(3):584-591.
[8] QUEREDA J J,DUSSURGET O,NAHORI M A,et al.Bacteriocin from epidemic Listeria strains alters the host intestinal microbiota to favor infection[J].Proceedings of the National Academy of Sciences ofthe United States of America, 2016,113(20):5706-5711.
[9] KOMMINENI S,BRETL D J,LAM V,et al.Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract[J].Nature,2015,526(7575):719-722.
[10] QUEREDA J J,MEZA-TORRES J,COSSART P,et al.Listeriolysin S:A bacteriocin from epidemic Listeria monocytogenes strains that targets the gut microbiota[J].Gut Microbes,2017,8(4):384-391.
[11] CLAYTON E M,DALY K M,GUINANE C M,et al.Atypical Listeria innocua strains possess an intact LIPI-3[J].BMC Microbiology,2014,14(1):1-9.
[12] COTTER P D,DRAPER L A,LAWTON E M,et al.Listeriolysin S,a novel peptide haemolysin associatedwith a subset of lineage Ⅰ Listeria monocytogenes[J].PLoS Pathogens,2008,4(9):e1000144.
[13] QUEREDA J J,NAHORI M A,MEZATORRES J,et al.Listeriolysin S is a streptolysin S-like virulence factor that targets exclusively prokaryotic cells in vivo[J].Mbio,2017,8(2):e00259-17.
[14] 陈云飞.单增李斯特菌ΔInlAB、ΔInlABC突变株的构建及部分生物学特性研究[D].石河子:石河子大学,2015. CHEN Y F.Construction and partial biological characteristics of Listeria monocytogenes ΔInlAB,ΔInlABC mutant strains[D].Shihezi:Shihezi University,2015.(in Chinese)
[15] 谢堃,乔军,孟庆玲,等.单核细胞增生李斯特菌非编码RNA rli87基因缺失株的构建、鉴定及其生长特性初步研究[J].畜牧兽医学报,2015,46(6):998-1003. XIE K,QIAO J,MENG Q L,et al.Construction,identification and growth characteristics of non-codingrli87 gene deletion strain of Listeria monocytogenes[J].Chinese Journal of Animal Husbandry and Veterinary Medicine,2015,46(6):998-1003.(in Chinese)
[16] 朱艳蕾.细菌生长曲线测定实验方法的研究[J].微生物学杂志,2016,36(5):108-112. ZHU Y L.Study on the method of determining the growth curve of bacteria[J].Journal of Microbiology,2016,36(5):108-112.(in Chinese)
[17] HERNANDEZMILIAN A,PAYERASCIFRE A.What is new inlisteriosis?[J].Biomed Research International,2014,2014:358051.
[18] COSSART P,TOLEDOARANA A.Listeria monocytogenes,a unique model in infection biology:An overview[J].Microbes & Infection,2008,10(9):1041-1050.
[19] 祝仁发,叶长芸.单核细胞增生李斯特菌的毒力因子[J].中国食品卫生杂志,2007,19(2):158-162. ZHU R F,YE C Y.Virulence factor of Listeria monocytogenes[J].Chinese Journal of Food Hygiene,2007,19(2):158-162.(in Chinese)
[20] 郎需龙,王兴龙.李斯特菌毒力因子及其调控机制的研究进展[J].中国畜牧兽医,2011,38(3):195-198. LANG X L,WANG X L.Advances in research on virulence factors and regulation mechanisms[J].China Animal Husbandry & Veterinary Medicine,2011,38(3):195-198.(in Chinese)
[21] 陈凤霞,叶精精,姜莉,等.单核细胞增生李斯特菌溶血素hly基因缺失株和回补株的构建[J].动物医学进展,2018,39(3):9-14. CHEN F X,YE J J,JIANG L,et al.Construction of Listeria monocytogenes listeriolysin O encoding gene (hly) deletion and complemented strains[J].Progressin Veterinary Medicine,2018,39(3):9-14.(in Chinese)
[22] QUEREDA J J,ANDERSSON C,COSSART P,et al.Role in virulence of phospholipases,listeriolysin O and listeriolysin S from epidemic Listeria monocytogenes using the chicken embryo infection model[J].Veterinary Research,2018,49(1):13-22.
[23] 崔焕忠,乔立桥,王义冲.单核细胞增生性李斯特菌的主要毒力因子及其致病机理[J].中国畜牧兽医,2010,37(1):128-133. CUI H Z,QIAO L Q,WANG Y C.Main virulence factors and pathogenicity of Listeria monocyto-genes[J].China Animal Husbandry & Veterinary Medicine,2010,37(1):128-133.(in Chinese)
[24] 李敏,杨谦.一种高效构建同源重组DNA片段的方法——融合PCR[J].中国生物工程杂志,2007,27(8):53-58. LI M,YANG Q.A highly efficient method for constructing homologous recombinant DNA fragments-Fusion PCR[J].Chinese Journal of Bioengineering,2007,27(8):53-58.(in Chinese)