单核细胞增多性李斯特菌弱毒株的生物学特性鉴定

doi:10.11843/j.issn.0366-6964.2014.02.016

畜牧兽医学报 ›› 2014, Vol. 45 ›› Issue (2): 274-280.doi: 10.11843/j.issn.0366-6964.2014.02.016

单核细胞增多性李斯特菌弱毒株的生物学特性鉴定

江玲丽¹,陈健舜²,李爱云³,万婧¹,周向阳¹,钱国英^4*,李彩燕⁴,方维焕⁵

（1.舟山出入境检验检疫局，舟山 316021；2.浙江省水产技术推广总站，杭州 310012；3.浙江大学医学院附属妇产科医院，杭州 310006； 4.浙江万里学院，宁波 315100；5.浙江大学动物预防医学研究所，杭州 310058）

收稿日期:2013-07-01 出版日期:2014-02-23 发布日期:2014-03-07
通讯作者: 钱国英，教授， E-mail：qiangy@zwu.edu.cn
作者简介:江玲丽（1980-），女，浙江温岭人，兽医师，博士，主要从事微生物与食品安全研究，E-mail：jll@zs.ziq.gov.cn
基金资助:
浙江万里学院省重中之重学科开放基金资助项目（KF2012007）；国家自然科学基金青年基金项目（30700592）；舟山群岛“千人计划”项目

Characterization of Listeria monocytogenes Strains with Low Virulence

JIANG Ling-li¹，CHEN Jian-shun²，LI Ai-yun³，WAN Jing¹，ZHOU Xiang-yang¹，QIAN Guo-ying^4*，LI Cai-yan⁴，FANG Wei-huan⁵

(1.Zhoushan EntryExit Inspection and Quarantine Bureau，Zhoushan 316021， China; 2.Zhejiang Fisheries Technical Extension Center，Hangzhou 310012，China; 3.Women′s Hospital，Zhejiang University School of Medicine，Hangzhou 310006， China; 4.Zhejiang Wanli University，Ningbo 315100， China；5.Institute of Preventive Veterinary Medicine，Zhejiang University，Hangzhou 310058， China)

Received:2013-07-01 Online:2014-02-23 Published:2014-03-07

摘要/Abstract

摘要：

拟探明单核细胞增多性李斯特菌（单增李斯特菌）天然弱毒株表型与基因型特征。针对弱毒株H4、54006，进行血清谱系分析、小鼠毒力测定、溶脂活性、细胞黏附力、细胞毒性、主要毒力基因检测及其mRNA转录水平测定等。结果表明菌株H4、54006为4a型谱系Ⅲ菌株，具有强溶脂活性、高细胞黏附率，但对小鼠毒力较弱；对HeLa细胞毒性显著高于其他菌株（P<0.05）。H4菌株主要毒力基因转录水平显著高于其他菌株（P<0.05）。序列分析发现2菌株主要毒力基因较保守，转录调控因子PrfA第145位由甘氨酸（G）突变为丝氨酸（S）。将丝氨酸回复突变为甘氨酸，H4菌株强溶脂活性消失，毒力增强2.18对数倍数，主要毒力基因转录水平降低。单增李斯特菌H4、54006弱毒可能与PrfA G145S突变有关，致细菌对宿主细胞毒性增强而从细胞内逸出，使其不能躲避宿主免疫系统而被清除。

关键词: 单增李斯特菌, 毒力, 细胞毒性, 转录调控因子PrfA

Abstract:

Phenotypic and genetic characteristics of two Listeria monocytogenes strains H4 and 54006 with naturally low pathogenicity were carried out to elucidate the possible molecular mechanisms.The two strains，together with other 3 L.monocytogenes strains representing different serovars，were investigated by serotype and lineage identification，virulence to mice，phospholipase activity，adhesion and cytotoxicity assay against HeLa cells，detection of virulence associated genes as well as their transcription levels via real-time PCR.Result were as follows：Strains H4 and 54006 belonged to serovar 4a and lineage Ⅲ，with notable phospholipase activity and increased adhesion to HeLa cells.However，these two strains had decreased pathogenicity to mice，but exhibited significantly higher cytotoxicity against HeLa cells （P<0.05）.The virulence associated genes in the two strains were quite homologous to other test strains，with PrfA depicting G145S mutation in strains H4 and 54006.The transcription levels of main virulence genes in strain H4 were significantly higher than those of other strains.Mutant strain H4-ΔprfA (substitution at position 145 from serine to glycine) was constructed by sitedirected mutagenesis and homologous recombination，with abolished phospholipase C activity and increased virulence to mice as compared with its parent strain H4 (10^5.00 vs 10^7.18) .The transcription levels of main virulence genes in mutant strain H4-ΔprfA were lower than those of the wild type strain.The study presents some clues that the low virulence of strains H4 and 54006 maybe result from PrfA G145S mutation，and leading to over-expression of PrfA regulated proteins.Hence，the strains might be exposed to the host immune responses with their stronger cytotoxicity.

Key words: Listeria monocytogenes, virulence, cytotoxicity, PrfA

中图分类号:

R378.99.4

江玲丽,陈健舜,李爱云,万婧,周向阳,钱国英,李彩燕,方维焕. 单核细胞增多性李斯特菌弱毒株的生物学特性鉴定[J]. 畜牧兽医学报, 2014, 45(2): 274-280.

JIANG Ling-li，CHEN Jian-shun，LI Ai-yun，WAN Jing，ZHOU Xiang-yang，QIAN Guo-ying，LI Cai-yan，FANG Wei-huan. Characterization of Listeria monocytogenes Strains with Low Virulence[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(2): 274-280.

参考文献

［1］ALLERBERGER F，WAGNER M.Listeriosis：a resurgent foodborne infection［J］.Clin Microbiol Infect，2010，16(1):16-23.
［2］Centers for Disease Control and Prevention.Listeriosis (Listeria infection) ［DB］.http：//www.cdc.gov/listeria/index.html［2012-05-28］.
［3］World Health Organization.Risk assessment of Listeria monocytogenes in ready-to-eat foods［DB］.http：//www.who.int/foodsafety/publications/ micro/en/mra4.pdf［2012-05-28］.
［4］ORSI R H，DEN BAKKER H C，WIEDMANN M.Listeria monocytogenes lineages：Genomics，evolution，ecology，and phenotypic characteristics［J］.Int J Med Microbiol，2011，301(2):79-96.
［5］CHEN J，ZHANG X，MEI L，et al.Prevalence of Listeria in Chinese food products from 13 provinces between 2000 and 2007 and virulence characterization of Listeria monocytogenes isolates［J］.Foodborne Pathog Dis，2009，6(1):7-14.
［6］VITULLO M，GRANT K A，SAMMARCO M L，et al.Real-time PCRs assay for serogrouping Listeria monocytogenes and differentiation from other Listeria spp［J］.Mol Cell Probes，2013，27(1):68-70.
［7］DESHAYES C，BIELECKA M K，CAIN R J，et al.Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo［J］.Mol Microbiol，2012，85(3):461-477.
［8］HAIN T，STEINWEG C，CHAKRABORTY T.Comparative and functional genomics of Listeria spp［J］.J Biotechol，2006，126(1)：37-51.
［9］CHEN J，LUO X，JIANG L，et al.Molecular characteristics and virulence potential of Listeria monocytogenes isolates from Chinese food systems［J］.Food Microbiol，2009，26(1):103-111.
［10］JIANG L，CHEN J，XU J，et al.Virulence characterization and genotypic analyses of Listeria monocytogenes isolates from food and processing environments in eastern China［J］.Int J Food Microbiol，2008，121(1):53-59.
［11］ZENG H，ZHANG X，SUN Z，et al.Multiplex PCR identification of Listeria monocytogenes isolates from milk and milk-processing environments［J］.J Sci Food Agri，2006，86(3):367-371.
［12］JIANG L L，XU J J，CHEN N，et al.Virulence phenotyping and molecular characterization of a low-pathogenicity isolate of Listeria monocytogenes from cow's milk［J］.Acta Biochim Biophys Sin (Shanghai)，2006，38(4):262-270.
［13］BAI F，CHEN J，JIANG L，et al.A single substitution in 5'-untranslated region of plcB is involved in enhanced broad-range phospholipase C activity in Listeria monocytogenes strain H4［J］.Acta Biochim Biophys Sin (Shanghai)，2011，43(4):275-283.
［14］VEGA Y，RAUCH M，BANFIELD M J，et al.New Listeria monocytogenes prfA* mutants，transcriptional properties of PrfA* proteins and structure-function of the virulence regulator PrfA［J］.Mol Microbiol，2004，52(6)：1553-1565.
［15］XAYARATH B，FREITAG N.Optimizing the balance between host and environmental survival skills：lessons learned from Listeria monocytogenes［J］.Future Microbiol, 2012，7(7):839-852.
［16］DE LAS HERAS A，CAIN R J，BIELECKA M K，et al.Regulation of Listeria virulence：PrfA master and commander［J］.Curr Opin Microbiol，2011，14(2)：118-127.
［17］DECATUR A，PORTNOY D A.A PEST-like sequence in Listeriolysin O essential for Listeria monocytogenes pathogenicity［J］.Science，2000，290(5493)：992-995.
［18］GEESE M，LOUREIRO J J，BEAR J E，et al.Contribution of Ena/VASP proteins to intracellular motility of Listeria requires phosphorylation and prolinerich core but not F-actin binding or multimerization［J］.Mol Biol Cell，2002，13(7)：2383-2396.

[1]	彭忠, 梁婉, 艾伟诚, 王斐, 华琳, 汤细彪, 陈焕春, 吴斌. 我国猪群中多杀性巴氏杆菌的基因型分析[J]. 畜牧兽医学报, 2019, 50(5): 1064-1072.
[2]	彭忠, 梁婉, 艾伟诚, 王斐, 华琳, 汤细彪, 陈焕春, 吴斌. 我国猪群中多杀性巴氏杆菌的基因型分析[J]. 畜牧兽医学报, 2019, 50(5): 1064-1072.
[3]	马维武, 周学章. 金黄色葡萄球菌PV-杀白细胞素的表达及其对奶牛乳腺上皮细胞的损伤作用[J]. 畜牧兽医学报, 2019, 50(10): 2105-2112.
[4]	陈小云, 刘莹, 薛麒, 朱真, 李启红, 印春生, 姚文生, 康凯, 杜吉革. 重组腐败梭菌α毒素对兔的免疫效力分析[J]. 畜牧兽医学报, 2018, 49(8): 1727-1734.
[5]	陈小云, 刘莹, 薛麒, 朱真, 李启红, 印春生, 姚文生, 康凯, 杜吉革. 重组腐败梭菌α毒素对兔的免疫效力分析[J]. 畜牧兽医学报, 2018, 49(8): 1727-1734.
[6]	杜吉革, 朱真, 薛麒, 李启红, 印春生, 彭小兵, 姚文生, 康凯, 陈小云. 产气荚膜梭菌重组ε毒素突变体的免疫保护力评价[J]. 畜牧兽医学报, 2018, 49(4): 777-785.
[7]	杜吉革, 朱真, 薛麒, 李启红, 印春生, 彭小兵, 姚文生, 康凯, 陈小云. 产气荚膜梭菌重组ε毒素突变体的免疫保护力评价[J]. 畜牧兽医学报, 2018, 49(4): 777-785.
[8]	王金玉, 姜晓文, 王娜, 王婕然, 于苗苗, 钟秋, 于文会. 穿心莲内酯影响猪链球菌生物被膜形成[J]. 畜牧兽医学报, 2018, 49(3): 629-636.
[9]	王金玉, 姜晓文, 王娜, 王婕然, 于苗苗, 钟秋, 于文会. 穿心莲内酯影响猪链球菌生物被膜形成[J]. 畜牧兽医学报, 2018, 49(3): 629-636.
[10]	孟丹, 孟庆玲, 乔军, 蔡扩军, 王登峰, 马帅, 伍晔晖, 才学鹏. 奶牛源金黄色葡萄球菌新疆流行株的耐药特性、毒力基因及分子分型[J]. 畜牧兽医学报, 2018, 49(1): 181-194.
[11]	孟丹, 孟庆玲, 乔军, 蔡扩军, 王登峰, 马帅, 伍晔晖, 才学鹏. 奶牛源金黄色葡萄球菌新疆流行株的耐药特性、毒力基因及分子分型[J]. 畜牧兽医学报, 2018, 49(1): 181-194.
[12]	曹喜锋, 秦伟森, 熊永晅, 李楠, 邓衔柏. 伏马毒素B₁对人脐静脉血管内皮细胞的毒性作用研究[J]. 畜牧兽医学报, 2017, 48(9): 1777-1784.
[13]	曹喜锋, 秦伟森, 熊永晅, 李楠, 邓衔柏. 伏马毒素B₁对人脐静脉血管内皮细胞的毒性作用研究[J]. 畜牧兽医学报, 2017, 48(9): 1777-1784.
[14]	郑亚妮, 高修歌, 刘晓晓, 滕佩, 季辉, 江善祥. 盐霉素对鸡原代心肌细胞的毒性作用及其机制的初步研究[J]. 畜牧兽医学报, 2017, 48(4): 740-751.
[15]	郑亚妮, 高修歌, 刘晓晓, 滕佩, 季辉, 江善祥. 盐霉素对鸡原代心肌细胞的毒性作用及其机制的初步研究[J]. 畜牧兽医学报, 2017, 48(4): 740-751.

单核细胞增多性李斯特菌弱毒株的生物学特性鉴定

Characterization of Listeria monocytogenes Strains with Low Virulence

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价