食源性单增李斯特菌inlA/inlB/inlC基因缺失株的构建及其生物学特性分析

doi:10.16431/j.cnki.1671-7236.2019.01.006

摘要/Abstract

摘要：

为探究内化素inlA/inlB/inlC基因对单增李斯特菌（Listeria monocytogenes，Lm）生物学特性的影响，本研究采用融合PCR方法构建Lm681 inlC基因缺失突变体，并构建pKSV7-△inlC穿梭载体，将其转化Lm681-△inlAB感受态细胞，利用温度（42 ℃）和氯霉素（10 μg/mL）抗性双重压力来实现同源重组，筛选同源重组子进行鉴定并研究其部分生物学特性。结果显示，PCR和测序结果证实成功构建了3基因缺失株（Lm681-△inlABC），且缺失株的生长特性与野生株相比无明显差异，溶血特性与野生株保持一致；小鼠感染试验显示，野生株Lm681、Lm681-△inlAB和Lm681-△inlABC对小鼠的致死率分别为80%（8/10）、60%（6/10）和40%（4/10），对小鼠的LD₅₀分别为4.36×10⁴、1.35×10⁶和2.95×10⁷ CFU，且Lm681-△inlABC在肝脏、脾脏及脑组织中的定植能力极显著低于野生株（P < 0.01）。研究结果表明，inlA/inlB/inlC基因对Lm致病性发挥具有一定的作用，为深入研究inlX基因介导Lm入侵宿主细胞过程中的作用机制提供了科学依据。

关键词: 单增李斯特菌; 内化素基因; 基因突变株; 生物学特性

Abstract:

In order to evaluate the effect of inlA/inlB/inlC genes on biological characteristics of Lm,the inlC gene deletion mutant was constructed by fusion PCR method,and then pKSV7-△inlC shuttle vector was constructed,which was transformed into competent cells Lm681-△inlAB.Homologous recombination was conducted using temperature (42℃)and chloramphenicol (10 μg/mL) resistance to achieve the inlC gene deletion strain,and then the homologous recombination was screened for identification and some biological characteristics were studied.PCR and sequencing results confirmed that Lm681-△inlABC was successfully obtained.The results showed that the growth characteristics of the deletion mutants were the same as the wild-type strain Lm681 and there was no significant difference.The hemolysis activity of the mutant strains were consistent with wild-type strain Lm681.In the mouse infection experiment,the mortality rate of wild-type strain Lm681,Lm681-△inlAB and Lm681-△inlABC were 80%(8/10),60%(6/10) and 40%(4/10),respectively.Results of mice virulence determination showed that the median lethal dose of wild-type strain,Lm681-△inlAB and Lm681-△inlABC were 4.36×10⁴,1.35×10⁶ and 2.95×10⁷ CFU,respectively.Furthermore,the colonization ability of the inlA/inlB/inlC genes deleted strain was extremely significantly lower than wild-type strain in liver,spleen and brain (P < 0.01).Collectively,these results indicated that inlA/inlB/inlC genes might play an important role in pathogenicity of Lm infection and provided scientific basis for the study on the mechanism in host cell invasion by Lm.

Key words: Listeria monocytogenes; internalin gene; gene mutant; biological characteristics

中图分类号:

S852.61

任静静, 杨铭伟, 陈云飞, 尹华, 王欣雨, 李蓓蓓, 蒋建军, 王鹏雁. 食源性单增李斯特菌inlA/inlB/inlC基因缺失株的构建及其生物学特性分析[J]. 《中国畜牧兽医》, 2019, 46(1): 45-53.

REN Jingjing, YANG Mingwei, CHEN Yunfei, YIN Hua, WANG Xinyu, LI Beibei, JIANG Jianjun, WANG Pengyan. Construction and Characterization of inlA/inlB/inlC Genes Deleted Mutant Strain of Food-borne Listeria monocytogenes[J]. , 2019, 46(1): 45-53.

参考文献

[1] BUCHANAN R L,GORRIS L G M,HAYMAN M M,et al.A review of Listeria monocytogenes:An update on outbreaks,virulence,dose-response,ecology,and risk assessments[J].Food Control,2017,75:1-13.
[2] LIANOU A,MOSCHONAS G,NYCHAS G J E,et al.Growth of Listeria monocytogenes in pasteurized vanilla cream pudding as affected by storage temperature and the presence of cinnamon extract[J].Food Research International,2018,106:1114-1122.
[3] ELMALI M,CAN H Y,YAMAN H.Prevalence of Listeria monocytogenes in poultry meat[J].Food Science and Technology,2015,35(4):672-675.
[4] PAGLIANO P,ASCIONE T,BOCCIA G,et al.Listeria monocytogenes meningitis in the elderly:Epidemiological,clinical and therapeutic findings[J].Le Infezioni in Medicina,2016,24(2):105-111.
[5] VASANTHAKRISHNAN R B,DE LAS HERAS A,SCORTTI M,et al.PrfA regulation offsets the cost of Listeria virulence outside the host[J].Environmental Microbiology,2015,17(11):4566-4579.
[6] COSSART P,LEBRETON A.A trip in the "New Microbiology" with the bacterial pathogen Listeria monocytogenes[J].FEBS Letters,2014,588(15):2437-2445.
[7] 任静静,杨铭伟,剡根强,等.环境因子及接种量对单增李斯特菌生长的影响[J].中国畜牧兽医,2017,44(5):1491-1497. REN J J,YANG M W,YAN G Q,et al.Effect of environmental factors and different inoculum size on growth state of Listeria monocytogenes[J].China Animal Husbandry & Veterinary Medicine,2017,44(5):1491-1497.(in Chinese)
[8] GRVNDLER T,QUEDNAU N,STUMP C,et al.The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier[J].Microbes & Infection,2013,15(4):291-301.
[9] BOHNE J,KESTLER H,UEBELE C,et al.Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA[J].Molecular Microbiology,2010,20(6):1189-1198.
[10] RAJABIAN T,GAVICHERLA B,HEISIG M,et al.The bacterial virulence factor InlC perturbs apical celljunctions and promotes cell-to-cell spread of Listeria[J].Nature Cell Biology,2009,11(10):1212-1218.
[11] GOUIN E,ADIB-CONQUY M,BALESTRINO D,et al.The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the IκB kinase subunit IKKα[J].Proceedings of the National Academy of Sciences of the United States of America,2010,107(40):17333-17338.
[12] BIERNE H,SABET C,PERSONNIC N,et al.Internalins:A complex family of leucine-rich repeat-containing proteins in Listeria monocytogenes[J].Microbes & Infection,2007,9(10):1156-1166.
[13] LUO Q,RAUCH M,MARR A K,et al.In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA-dependent and independent promoters that are differentially activated by GTP[J].Molecular Microbiology,2004,52(1):39-52.
[14] 白春光,殷月兰,贾艳艳,等.单核细胞增生性李斯特菌prfA基因缺失株的构建及其生物学特性[J].微生物学报,2011,51(11):1555-1560. BAI C G,YIN Y L,JIA Y Y,et al.Construction and characterization of Listeria monocytogenes ΔprfA mutant strains[J].Acta Microbiologica Sinica,2011,51(11):1555-1560.(in Chinese)
[15] 任静静,杨铭伟,陈云飞,等.单增李斯特菌inlA/inlB双基因缺失株生长特性观察及对小鼠致病性研究[J].中国人兽共患病学报,2016,32(6):529-534. REN J J,YANG M W,CHEN Y F,et al.Biological characters and pathogenicity of Listeria monocytogenes △inlA/△inlB double gene deletion strains[J].Chinese Journal of Zoonoses,2016,32(6):529-534.(in Chinese)
[16] DAISUKE K,HAJIME T,SATOKO M,et al.Comparison of the major virulence-related genes of Listeria monocytogenes in internalin A truncated strain 36-25-1 and a clinical wild-type strain[J].BMC Microbiology,2014,14(1):1-7.
[17] BERGMANN B,RAFTELSBAUER D,KUHN M,et al.InlA-but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins[J].Molecular Microbiology,2002,43(3):557-570.
[18] HAMON M,BIERNE H,COSSART P.Listeria monocytogenes:A multifaceted model[J].Nature Reviews Microbiology,2006,4(6):423-434.
[19] GHEBREHIWET B,JESTY J,PEERSCHKE E I.gClq-R/p33:Structure function predicitions from the crystal structure[J].Immunobiology,2002,205(4-5):421-432.
[20] LEUNG N,GIANFELICE A,GRAYOWEN S D,et al.Impact of the Listeria monocytogenes protein InlC on infection in mice[J].Infection & Immunity,2013,81(4):1334-1340.
[21] 任静静,杨铭伟,王鹏雁,等.单增李斯特菌(LM90)inlA单基因及inlB/inlA双基因缺失株的构建及其生物学特性初步研究[J].中国预防兽医学报,2018,40(4):294-300. REN J J,YANG M W,WANG P Y,et al.Construction and characterization of LM90-△inlA and LM90-△inlB/△inlA mutant strains of Listeria monocytogenes[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(4):294-300.(in Chinese)
[22] 魏丽,邓兴梅,蒋建军,等.单增李斯特菌inlB基因缺失株的构建及其生物学特性[J].动物医学进展,2014,35(10):19-24. WEI L,DENG X M,JIANG J J,et al.Construction and characterization of Listeria monocytogenes strain with inlB gene deletion[J].Progress in Veterinary Medicine,2014,35(10):19-24.(in Chinese)
[23] 刘武康,陈国薇,吴嫚,等.inlA和inlB基因缺失对单核细胞增生性李斯特菌侵袭HT29结肠癌细胞的影响[J].食品科学,2016,37(23):166-172. LIU W K,CHEN G W,WU M,et al.Effect of mutations in the inlA and inlB genes on the invasion ofListeria monocytogenes to HT29 conlon cancer cells[J].Food Science,2016,37(23):166-172.(in Chinese)
[24] BONAZZI M,VEIGA E,PIZARROCERDÁ J,et al.Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes[J].Cellular Microbiology,2008,10(11):2208-2222.
[25] COSSART P,TOLEDO-ARANA A.Listeria monocytogenes,a unique model in infection biology:An overview[J].Microbes & Infection,2008,10(9):1041-1050.