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| LIPI-4 EII对单核细胞增生性李斯特菌关键毒力因子转录调控的影响 |
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史唯地,马勋,刘彩霞,寇丽君,吕双飞,任慧杰,曾东东,王静*,孔翠莲,高盛杰,钱瑞宣
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| (石河子大学 动物科技学院, 新疆 石河子 832000) |
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| 摘要: |
| 为探究单核细胞增生性李斯特菌(LM)毒力岛4(LIPI-4)中膜透性酶(EII)对LM毒力基因表达的调控作用，本研究分别构建EII缺失株(LM928ΔEII)、强启动子回补株(CLM928ΔEII-Phelp)和天然启动子回补株(CLM928ΔEII-Pnative)，测定各菌株在体外培养中的生长曲线和在永生化人脑微血管内皮细胞(HCMEC/D3)内的增殖情况；RT-qPCR检测体外培养和HCMEC/D3细胞内各菌株9个关键毒力基因的转录水平。结果显示:1)EII基因缺失株LM928ΔEII构建成功，重组回补质粒pIMK2-EII和pIMK2-EII-Pnative及回补株CLM928ΔEII-Phelp和CLM928ΔEII-Pnative构建成功。2)在体外培养下，EII对LM的生长曲线没有影响。但在侵染HCMEC/D3后，LM928ΔEII在8和12 h的胞内细菌量显著高于LM928(P<0.05)，2和4 h的胞内细菌量极显著高于LM928(P<0.01)，CLM928ΔEII-Pnative在2、4和6 h恢复至野生株水平(P>0.05)，而CLM928ΔEII-Phelp在2、4、6、8、10和12 h均极显著低于野生株(P<0.01)。3)体外培养条件下，相比于野生株，LM928ΔEII中66.7%(6/9)的毒力基因转录水平极显著上调(P<0.01)；CLM928ΔEII-Phelp中66.7%(6/9)的基因转录水平上升1~4倍，CLM928ΔEII-Pnative中88.9%(8/9)的基因转录水平倍数变化在1倍以内。在HCMEC/D3细胞内，相比于野生株，LM928ΔEII中66.7%(6/9)的毒力基因转录水平极显著上调(P<0.01)；CLM928ΔEII-Phelp中33.3%(3/9)的基因转录水平上调1~3倍，CLM928ΔEII-Pnative中基因转录水平倍数变化在1倍以内。综上，LIPI-4 EII对LM关键毒力因子的转录具有负调控作用。本研究为系统探究LIPI-4参与LM致病性机制奠定基础。 |
| 关键词: 单核细胞增生性李斯特菌 LIPI-4 膜透性酶(EII) 缺失株 毒力基因 |
| DOI：10.11841/j.issn.1007-4333.2023.08.16 |
| 投稿时间：2022-11-11 |
| 基金项目:国家自然科学基金资助项目(32160834,32160833)；省部共建绵羊遗传改良与健康养殖国家重点实验室开放课题(MYSKLKF201905)；动物疾病防控兵团重点实验室开放课题(2020BTDJ05) |
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| Transcriptional regulation of key virulence factors in Listeria monocytogenes by EII of LIPI-4 |
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SHI Weidi,MA Xun,LIU Caixia,KOU Lijun,LV Shuangfei,REN Huijie,ZENG Dongdong,WANG Jing*,KONG Cuilian,GAO Shengjie,QIAN Ruixuan
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| (College of Animal Science and Technology, Shihezi University, Xinjiang Shihezi 832000, China) |
| Abstract: |
| To explore the regulatory effect of membrane permeability enzyme(EII)of Listeria monocytogenes(LM)pathogenicity island 4(LIPI-4)on the expression of LM virulence genes, in the study, the EII deletion mutant(LM928ΔEII), strong promoter complemented strain(CLM928ΔEII-Phelp)and native promoter complemented strain(CLM928ΔEII-Pnative)were constructed, respectively, and the growth curve in vitro and intracellular proliferation in HCMEC/D3 of each strain were measured. The transcription levels of 9 key virulence genes in each strain were detected by RT-qPCR in vitro and HCMEC/D3 cells. The results showed as follows: 1)EII gene deletion mutant LM928ΔEII was successfully constructed. The recombinant complemented plasmid pIMK2-EII and pIMK2-EII-Pnative, complemented strain CLM928ΔEII-Phelp and CLM928ΔEII-Pnative were successfully constructed. 2)In vitro, EII had no effect on the growth curve of LM. The number of proliferation of LM928ΔEII in HCMEC/D3 cells was significantly higher than that of LM928 at 8 and 12 h(P<0. 05), and the amount of intracellular bacteria was extremely significantly higher than that of LM928 at 2, 4 h(P<0. 01). CLM928ΔEII-Pnative was restored to the level of wild type strain at 2, 4 and 6 h(P>0. 05), while CLM928ΔEII-Phelp was significantly lower than that of wild type strain at 2, 4, 6, 8, 10 and 12 h(P<0. 01). 3)In vitro, compared with the wild type strain, 66. 7%(6/9)of virulence gene transcription levels in LM928ΔEII were extremely significantly up-regulated(P<0. 01), CLM928ΔEII-Phelp increased the gene transcription levels of 66. 7%(6/9)by 1-4 times, while 88. 9%(8/9)gene transcription levels fold change of CLM928ΔEII-Pnative were less than 1 time. In HCMEC/D3 cells, compared with the wild type strain, 66. 7%(6/9)of virulence gene transcription levels in LM928ΔEII were extremely significantly up-regulated(P<0. 01), CLM928ΔEII-Phelp increased the gene transcription levels of 33. 3%(3/9)by 1-3 times while the gene transcription levels of CLM928ΔEII-Pnative were all less than 1 time. In conclusion, LIPI-4EII negatively regulates the transcription of key virulence factors of LM. This study laid the foundation for systematically exploring the mechanism of LIPI-4 involvement in LM pathogenicity. |
| Key words: Listeria monocytogenes LIPI-4 membrane permeability enzyme Ⅱ deletion mutants virulence gene |
