单增李斯特菌Lm4b_02324基因缺失株的构建及生物特性研究

doi:10.16431/j.cnki.1671-7236.2023.05.025

摘要/Abstract

摘要： 【目的】研究Lm4b_02324基因编码的6-磷酸-β-葡萄糖苷酶对单增李斯特菌生物学特性的影响,为研究单增李斯特菌的致病机制奠定基础。【方法】本研究使用重叠延伸PCR技术,并通过连续传代来构建稳定遗传野生型单增李斯特菌Lm928株的Lm4b_02324基因缺失株(Lm928Δm4b_02324)与Lm4b_02324基因回补株(CLm928Δm4b_02324)。通过检测不同培养时间的D_{600 nm}值并绘制生长曲线分析Lm928、Lm928ΔLm4b_02324和CLm928ΔLm4b_02324 3株菌的生长特性;对小鼠腹腔注射不同浓度的3株菌,观察感染后临床症状,统计死亡率,通过寇氏法计算3株菌对小鼠的半数致死量(LD₅₀);将Lm928、Lm928ΔLm4b_02324和CLm928ΔLm4b_02324以感染复数(multiplicity of infection,MOI)为10分别感染HCMEC细胞,通过检测感染后不同时间细胞内的细菌数量分析各菌株的黏附与侵袭能力和胞内生存能力;提取野生株与缺失株DNA,使用prfA、mpl、hly等9对毒力相关因子的引物,利用实时荧光定量PCR技术测定其表达情况。【结果】分别成功获得了大小为1 209 bp的基因缺失株Lm928ΔLm4b_02324(野生株:2 517 bp)和1 301 bp的回补株CLm928ΔLm4b_02324(野生株:1 301 bp,条带一致),且缺失株传至15代后未出现回复突变现象;生长曲线结果显示,野生株、缺失株和回补株的生长状况差异不显著(P＞0.05);LD₅₀测定结果显示,与野生型相比缺失株LD₅₀显著降低(P＜0.05);黏附与侵袭力试验结果显示,缺失株的胞内存活数显著或极显著高于野生株与回补株(P＜0.05; P＜0.01);感染HBMEC细胞12 h内,缺失株的胞内活菌数显著高于野生株 (P＜0.05)。实时荧光定量PCR结果显示,Lm4b_02324 基因的缺失使得prfA、inlA、plcA基因表达显著降低(P＜0.05);hly、actA、mpl、inlC基因表达极显著降低(P＜0.01)。【结论】本研究结果表明,Lm4b_02324基因缺失会导致单增李斯特菌对小鼠的致死率升高并增强其在胞内增殖能力,为研究Lm4b_02324基因在单增李斯特菌致病中的作用奠定基础。

关键词: 单增李斯特菌; 缺失株; 6-磷酸-β-葡萄糖苷酶; 胞内生存

Abstract: 【Objective】 This experiment was aimed to study the effects of 6-phospho-β-glucosidase encoded by the Lm4b_02324 gene on the biological characteristics of Listeria monocytogenes,and to lay a foundation for the study of the pathogenic mechanism of Listeria monocytogenes.【Method】 In this study,overlapping extended PCR technique was used to construct the Lm4b_02324 gene deletion strain (Lm928ΔLm4b_02324) and Lm4b_02324 gene complement strain (CLm928ΔLm4b_02324) of stable genetic wild type Lm928 through continuous passage.The growth characteristics of Lm928,Lm928ΔLm4b_02324 and CLm928ΔLm4b_02324 were analyzed by detecting D_{600 nm} values of different culture time and drawing growth curves.The mice were injected intraperitoneally with three strains of different concentrations,and the clinical symptoms after infection were observed.The lethal dose 50% (LD₅₀) of the three strains to the mice was calculated by Koch’s calculation method.Lm928,Lm928ΔLm4b_02324 and CLm928ΔLm4b_02324 were infected with multiplicity of infection (MOI) as 10,respectively.The adhesion and invasion ability and intracellular survival ability of each strain were analyzed by detecting the number of bacteria in cells at different time after infection.DNA of wild and deletion strains was extracted.Primers of 9 virulence related factors,such as prfA,mpl and hly,were used to detect their expressions by Real-time quantitative PCR.【Result】 PCR identification results showed that gene deletion strain Lm928ΔLm4b_02324 (wild strain:2 517 bp,deletion strain:1 209 bp) and the complement strain CLm928ΔLm4b_02324 (1 301 bp,consistent with the band of wild strain) were successfully obtained,and the deletion strain did not appear back mutation phenomenon after 15 generations.The results of growth curve showed that there was no significant difference in the growth status of wild strain,deletion strain and complement strain.LD₅₀ determination results showed that the LD₅₀ of the deletion strain was significantly lower than that of the wild strain (P＜0.05).The results of adhesion and invasion test showed that the intracellular survival number of the deletion strain was significantly or extremely significantly higher than that of wild strain and complement strain (P＜0.05 or P＜0.01).Within 12 h after infection with HBMEC cells,the intracellular viable bacteria number of the deletion strain was higher than that of wild strain,and the difference was significant (P＜0.05).Real-time quantitative PCR results showed that the deletion of Lm4b_02324 gene significantly decreased the expression of prfA,inlA and plcA genes(P＜0.05),and the expressions of hly,actA,mpl and inlC were extremely significantly decreased (P＜0.01).【Conclusion】 The results of this study showed that the deletion of Lm4b_02324 gene could increase the mortality of Listeria monocytogenes in mice and enhance its intracellular proliferation ability,which laid a foundation for the study of the role of Lm4b_02324 gene in the pathogenesis of Listeria monocytogenes.

Key words: Listeria monocytogenes; deletion strains; 6-phospho-β-glucosidase; intracellular survival

中图分类号:

S852.61

曾东东, 刘彩霞, 寇丽君, 秦赫, 晋瑞婕, 王静, 任静静, 马勋, 蒋建军. 单增李斯特菌Lm4b_02324基因缺失株的构建及生物特性研究[J]. 中国畜牧兽医, 2023, 50(5): 1981-1990.

ZENG Dongdong, LIU Caixia, KOU Lijun, QIN He, JIN Ruijie, WANG Jing, REN Jingjing, MA Xun, JIANG Jianjun. Construction and Biological Characteristics of Lm4b_02324 Gene Deletion Strains of Listeria monocytogenes[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1981-1990.

参考文献

[1] CHENG C,HAN X,XU J,et al.YjbH mediates the oxidative stress response and infection by regulating SpxA1 and the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) in Listeria monocytogenes[J].Gut Microbes,2021,13(1):1-19.
[2] GHOSH P,HALVORSEN E M,AMMENDOLIA D A,et al.Invasion of the brain by Listeria monocytogenes is mediated by InlF and host cell vimentin[J].mBio,2018,9(1):e00160-18.
[3] TIENSUU T,GUERREIRO D N,OLIVEIRA A H,et al.Flick of a switch:Regulatory mechanisms allowing Listeria monocytogenes to transition from a saprophyte to a killer[J].Microbiology(Reading,England),2019,165(8):819-833.
[4] DEMATRE N,VAN DAMME I,DE ZUTTER L,et al.Occurrence,distribution and diversity of Listeria monocytogenes contamination on beef and pig carcasses after slaughter[J].Meat Science,2020,169:108177.
[5] DESAI A N,ANYOHA A,MADOFF L C,et al.Changing epidemiology of Listeria monocytogenes outbreaks,sporadic cases,and recalls globally:A review of ProMED reports from 1996 to 2018[J].International Journal of Infectious Diseases,2019,84:48-53.
[6] RAMASWAMY V,CRESENCE V M,REJITHA J S,et al.Listeria-Review of epidemiology and pathogenesis[J]. Journal of Microbiology Immunology and Infection,2007,40(1):4-13.
[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] 王欣雨,尹华,任静静,等.单增李斯特菌llsB基因克隆及序列分析[J].中国畜牧兽医,2019,46(7):1890-1898. WANG X Y,YIN H,REN J J,et al.Cloning and sequence analysis of Listeria monocytogenes llsB gene[J].China Animal Husbandry&Veterinary Medicine,2019,46(7):1890-1898.(in Chinese)
[9] KING S J.Pneumococcal modification of host sugars:A major contributor to colonization of the human airway?[J].Molecular Oral Microbiology,2010,24(1):15-24.
[10] ALESSANDRO B,LAURA M,FRANCESCA D,et al.A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae[J].PLoS One,2017,7(3):e33320.
[11] TETTELIN H,NELSON K E,PAULSEN I T,et al.Complete genome sequence of a virulent isolate of Streptococcus pneumoniae[J].Science(New York,N.Y.),2001,293(5529):498-506.
[12] HELFERT C,GOTSCHE S,DAHL M K.Cleavage of trehalose-phosphate in Bacillus subtilis is catalysed by a phospho-alpha-(1-1)-glucosidase encoded by the treA gene[J].Molecular Microbiology,1995,16(1):111-120.
[13] COTE C K,HONEYMEN A L.The LicT protein acts as both a positive and a negative regulator of loci within the bgl regulon of Streptococcus mutans[J]. Microbiology(Reading,England),2003,149(Pt 5):1333-1340.
[14] VELDMAN W,LIBERATO M V,ALMEIDA V M,et al.X-ray structure,bioinformatics analysis,and substrate specificity of a 6-phospho-β-glucosidase glycoside hydrolase 1 enzyme from Bacillus licheniformis[J].Journal of Chemical Information and Modeling,2020,60(12):6392-6407.
[15] VELDMAN W,LIBERATO M V,SOUZA V P,et al.Differences in gluco and galacto substrate-binding interactions in a dual 6Pβ-glucosidase/6Pβ-galactosidase glycoside hydrolase 1 enzyme from Bacillus licheniformis[J].Journal of Chemical Information and Modeling,2021,61(9):4554-4570.
[16] MAURY M M,TSAI Y H,CHARLIER C,et al.Uncovering Listeria monocytogenes hypervirulence by harnessing its biodiversity[J]. Nature Genetics,2016,48(3):308-311.
[17] FIONA Z,COSETTE G,EMILIE L,et al.Listeria monocytogenes ability to survive desiccation:Influence of serotype,origin,virulence,and genotype[J].International Journal of Food Microbiology,2017,248:82-89.
[18] 江玲丽,高有领,周向阳,等.单增李斯特菌膜裂解相关基因缺失突变株的构建及生物学特性鉴定[J].畜牧兽医学报,2016,47(4):779-788. JIANG L L,GAO Y L,ZHOU X Y,et al.Construction and characterization of mutant strains from Listeria monocytogenes with deletion of vacuolar lysis related genes[J].Acta Veterinaria et Zootechinca Sinica,2016,47(4):779-788.(in Chinese)
[19] 杜冬冬,康立超,张奇文,等.单增李斯特菌lmo0331基因缺失株的构建及对环境耐受性的影响[J].动物医学进展,2020,41(5):12-18. DU D D,KANG L C,ZHANG Q W,et al.Construction and environmental tolerance of lmo0331 gene deletion mutant strain of Listeria monocytogenes[J].Progress in Veterinary Medicine,2020,41(5):12-18.(in Chinese)
[20] MCKESSAR S J,HAKENBECK R.The two-component regulatory system TCS08 is involved in cellobiose metabolism of Streptococcus pneumoniae R6[J].Journal of Bacteriology,2007,189(4):1342-1350.
[21] TERRA V S,ZHI X Y,KAHYA H F,et al.Pneumococcal 6-phospho-β-glucosidase (BglA3) is involved in virulence and nutrient metabolism[J].Infection and Immunity,2016,84(1):286-292.
[22] GAHAN C G,HILL C.The use of listeriolysin to identify in vivo induced genes in the gram-positive intracellular pathogen Listeria monocytogenes[J].Molecular Microbiology,2000,36(2):498-507.
[23] 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-ΔinlA and LM90-ΔinlB/ΔinlA mutant strains of Listeria monocytogenes[J]. Chinese Journal of Preventive Veterinary Medicine,2018,40(4):294-300.(in Chinese)
[24] 李巧巧,王欣雨,潘星羽,等.溶血素基因缺失对单增李斯特菌在小鼠肠道内定殖的影响[J].畜牧与兽医,2022,54(9):104-108. LI Q Q,WANG X Y,PAN X Y,et al.Effect of the hemolysin gene deletion on Listeria monocytogenes colonization in the intestinal tracts of mice[J].Animal Husbandry and Veterinary Medicine,2022,54(9):104-108.(in Chinese)
[25] 赵莹莹,贾艳艳,杜付玉,等.单增李斯特菌hly基因缺失株的构建及重要特性分析[J].中国预防兽医学报,2019,41(5):509-513. ZHAO Y Y,JIA Y Y,DU F Y,et al.Construction and characteristic analysis of Listeria monocytogenes listeriolysin O encoding gene (hly) deletion strain in mice[J]. Chinese Journal of Preventive Veterinary Medicine,2019,41(5):509-513.(in Chinese)
[26] 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 determinants and its evolution of the genus Listeria[J].Acta Microbiologica Sinica,2007,47(4):738-742.(in Chinese)
[27] SCHNUPF P,PORTNOY D A.Listeriolysin O:A phagosome-specific lysin[J]. Microbes and Infection,2007,9(10):1176-1187.
[28] AN C L,LIM W J,HONG S Y,et al.Structural and biochemical analysis of the asc operon encoding 6-phospho-β-glucosidase in Pectobacterium carotovorum subsp.carotovorum LY34[J].Research in Microbiology,2004,156(2):145-153.