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中国防痨杂志 ›› 2023, Vol. 45 ›› Issue (6): 566-574.doi: 10.19982/j.issn.1000-6621.20230059

• 论著 • 上一篇    下一篇

结核分枝杆菌Rv2333c在巨噬细胞内功能的初步研究

何萍1, 王怡婷1, 宋泽萱1, 夏辉2, 王胜芬2, 贺文从1, 郑扬2, 赵雁林2()   

  1. 1中国疾病预防控制中心传染病预防控制所,北京 102206
    2中国疾病预防控制中心结核病预防控制中心,北京 102206
  • 收稿日期:2023-03-01 出版日期:2023-06-10 发布日期:2023-06-06
  • 通信作者: 赵雁林 E-mail:zhaoyl@chinacdc.cn
  • 基金资助:
    国家重点研发计划(2022YFC2305200)

Preliminary study on the gene function of Mycobacterium tuberculosis Rv2333c in macrophages

He Ping1, Wang Yiting1, Song Zexuan1, Xia Hui2, Wang Shengfen2, He Wencong1, Zheng Yang2, Zhao Yanlin2()   

  1. 1National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
    2National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
  • Received:2023-03-01 Online:2023-06-10 Published:2023-06-06
  • Contact: Zhao Yanlin E-mail:zhaoyl@chinacdc.cn
  • Supported by:
    National Key R&D Program(2022YFC2305200)

摘要:

目的: 在耻垢分枝杆菌内过表达结核分枝杆菌Rv2333c,探究结核分枝杆菌Rv2333c在细菌侵染巨噬细胞过程中所发挥的功能。方法: 构建表达Rv2333c基因的重组质粒pSUM-MCS2-Rv2333c,进而得到过表达Rv2333c的耻垢分枝杆菌菌株;用空载菌株(Ms_Vec)和过表达菌株(Ms_Rv2333c)感染巨噬细胞,并在不同时间点收集样品。随后进行细菌胞内存活实验,检测乳酸脱氢酶(LDH)含量以分析Rv2333c对巨噬细胞毒性的影响;之后通过提取细胞样品的RNA、上清培养液和细胞沉淀裂解物,利用逆转录聚合酶链式反应(RT-PCR)、酶联免疫吸附测定(ELISA)和蛋白免疫印迹(Western blotting)等技术检测Rv2333c对巨噬细胞内相关细胞因子的分泌,以及核因子激活B细胞的κ-轻链增强(NF-κB)和磷酸化细胞外调节蛋白激酶1/2(p-ERK1/2)等通路的影响。结果: 实验成功构建了Rv2333c表达载体,并获得过表达Rv2333c的重组耻垢分枝杆菌。Ms_Rv2333c菌株感染巨噬细胞后,细胞毒性检测LDH结果显示:Rv2333c在侵染巨噬细胞早期(6h)即对宿主的毒性明显下降[Ms_Vec:(52.55±0.90)%,Ms_Rv2333c:(27.87±1.77)%;t=17.560,P<0.001],但不影响细菌胞内菌落形成单位(log10CFU)的增殖(Ms_Vec:7.45±0.05,Ms_Rv2333c:7.35±0.16;t=1.069,P=0.345)。RT-PCR结果显示:Rv2333c可使巨噬细胞内早期(6h)白细胞介素(IL)1β mRNA水平(2-ΔΔCt值)表达下调(Ms_Vec:657.43±20.96,Ms_Rv2333c:521.36±25.97;t=5.767,P=0.005),而在侵染后期(48h)则促进其表达上调(Ms_Vec:1548.53±125.17,Ms_Rv2333c:2168.13±130.00;t=4.855,P=0.008);但Rv2333c在感染早期(6h)和晚期(48h)均可使IL-6 mRNA表达上调(Ms_Vec:2.46±0.49和776.41±15.38,Ms_Rv2333c:6.02±0.37和1642.76±51.15;t值分别为8.234和22.940,P值分别为0.001和<0.001);同时也均上调γ-干扰素(IFN-γ)mRNA的表达(Ms_Vec:2.52±0.09和2.76±0.32,Ms_Rv2333c:17.24±0.47和8.52±0.08;t值分别为43.760和25.090,P值均<0.001)。Western blotting结果显示:Rv2333c可使巨噬细胞内NF-κB表达和ERK1/2磷酸化水平在3h时即明显上升[Ms_Vec:(1.00±0.14)%和(1.00±0.06)%,Ms_Rv2333c:(1.85±0.29)%和(1.74±0.03)%;t值分别为 3.765和15.040,P值分别为0.020和<0.001],至24h时仍明显上升[Ms_Vec:(1.00±0.14)%和(1.00±0.02)%,Ms_Rv2333c:(1.45±0.15)%和(1.17±0.04)%;t值分别为3.041和5.538,P值分别为0.038和0.005]。结论: Rv2333c在结核分枝杆菌致病过程中起到非常重要的作用。在结核分枝杆菌侵染巨噬细胞早期Rv2333c即可发挥抑制细胞凋亡的作用,有助于细菌在胞内存活。但在侵染中后期,Rv2333c可通过促进巨噬细胞炎症因子mRNA的表达诱导细胞发生炎症反应并参与其中。还可通过影响ERK1/2的磷酸化间接参与NF-κB转移进入细胞核及调控转录因子的活动。

关键词: 巨噬细胞, 分枝杆菌,结核, 基因表达调控, 炎症趋化因子类, 基因,Rv2333c

Abstract:

Objective: To explore the gene function of Mycobacterium tuberculosis (MTB) Rv2333c by overexpressing Rv2333c in Mycolicibacterium (M.) smegmatis in the process of infected macrophages. Methods: Overexpression of Rv2333c in M.smegmatis was achieved based on the construction of pSUM-MCS2-Rv2333c. Samples were collected at different time points after infecting macrophages with the empty strain (Ms_Vec) or the overexpression strain (Ms_Rv2333c). Then, the bacterial intracellular viability was assessed by counting colony-forming units (CFU) and the cytotoxicity of Rv2333c to the cell was detected by measuring the lactate dehydrogenase (LDH) activity in the cell media. Total RNA was extracted and the supernatant of culture media and cell lysates was collected to investigate Rv2333c effect on cytokines secretion and NF-κB/MAPK pathway in macrophages through reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments. Results: Rv2333c of MTB was successfully expressed in M.smegmatis. The cytotoxicity significantly decreased at the early stage (6 h) of macrophages infected with Ms_Rv2333c (LDH: Ms_Vec: (52.55±0.90) %, Ms_Rv2333c: (27.87±1.77) %; t=17.560, P<0.001), but the intracellular proliferation of Ms_Rv2333c was not affected (log10CFU: Ms_Vec: 7.45±0.05, Ms_Rv2333c: 7.35±0.16; t=1.069, P=0.345). RT-PCR revealed that IL-1β mRNA was downregulated at the early stage (6 h, Ms_Vec: 657.43±20.96, Ms_Rv2333c: 521.36±25.97; t=5.767, P=0.005), but upregulated at the late stage of macrophages infected with Ms_Rv2333c (48 h, Ms_Vec: 1548.53±125.17, Ms_Rv2333c: 2168.13±130.00; t=4.855, P=0.008). However, IL-6 mRNA was upregulated at the early (6 h) and late stage (48 h) of macrophages infected with Ms_Rv2333c (Ms_Vec: 2.46±0.49 & 776.41±15.38, Ms_Rv2333c: 6.02±0.37 & 1642.76±51.15; t=8.234 & 22.940, P=0.001 & <0.001, respectively). And the interferon-γ (IFN-γ) mRNA was also upregulated (Ms_Vec: 2.52±0.09 & 2.76±0.32, Ms_Rv2333c: 17.24±0.47 & 8.52±0.08; t=43.760 & 25.090, all P<0.001, respectively). The results of Western blotting showed that the expression of NF-κB and phosphorylation of ERK1/2 significantly increased in macrophages 3 hours after infecting with Ms_Rv2333c and maintained at high level even at 24 h (3 h, Ms_Vec: (1.00±0.14) % & (1.00±0.06) %, Ms_Rv2333c: (1.85±0.29) % & (1.74±0.03) %; t=3.765 & 15.040, P=0.020 & <0.001, respectively; 24 h, Ms_Vec: (1.00±0.14) % & (1.00±0.02) %, Ms_Rv2333c: (1.45±0.15) % & (1.17±0.04) %; t=3.041 & 5.538, P=0.038 & 0.005, respectively). Conclusion: Rv2333c plays an important role in the pathogenesis of MTB. Rv2333c inhibited apoptosis at the early stage of infection, which is helpful for the survival of bacteria in macrophages. In addition, Rv2333c can promote the expression of inflammatory factors at the middle and late stages of infection to induce inflammatory reaction in cells. It can also indirectly participate in transferring of NF-κB to the nucleus and regulating the activity of transcription factors by affecting the ERK1/2 phosphorylation capacity.

Key words: Macrophages, Mycobacterium tuberculosis, Gene expression regulation, Chemokines, Gene, Rv2333c

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