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
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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(10μg/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4.36×10~4、1.35×10~6和2.95×10~7 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。
In order to evaluate the effect of inlA/inlB/inlCgenes on biological characteristics of Lm,the inlCgene 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(10μg/mL)resistance to achieve the inlCgene 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)and40%(4/10),respectively.Results of mice virulence determination showed that the median lethal dose of wild-type strain,Lm681-△inlAB and Lm681-△inlABC were 4.36×10`4,1.35×10~6 and 2.95×10~7 CFU,respectively.Furthermore,the colonization ability of the inlA/inlB/inlCgenes 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/inlCgenes 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.
In order to evaluate the effect of inlA/inlB/inlCgenes on biological characteristics of Lm,the inlCgene 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(10μg/mL)resistance to achieve the inlCgene 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)and40%(4/10),respectively.Results of mice virulence determination showed that the median lethal dose of wild-type strain,Lm681-△inlAB and Lm681-△inlABC were 4.36×10`4,1.35×10~6 and 2.95×10~7 CFU,respectively.Furthermore,the colonization ability of the inlA/inlB/inlCgenes 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/inlCgenes 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.
引文
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[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 monocytogenesΔprfA mutant strains[J].Acta Microbiologica Sinica,2011,51(11):1555-1560.(in Chinese)
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[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 of Listeria monocytogenes to HT29conlon cancer cells[J].Food Science,2016,37(23):166-172.(in Chinese)
[24]BONAZZI M,VEIGA E,PIZARROCERDJ,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.
[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 Listeriavirulence 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]GRNDLER 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 bloodcerebrospinal 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 cell junctions 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 inlCand 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 monocytogenesΔprfA 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/△inlBdouble 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 36-25-1and 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 of Listeria monocytogenes to HT29conlon cancer cells[J].Food Science,2016,37(23):166-172.(in Chinese)
[24]BONAZZI M,VEIGA E,PIZARROCERDJ,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.