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中国防痨杂志 ›› 2018, Vol. 40 ›› Issue (6): 616-621.doi: 10.3969/j.issn.1000-6621.2018.06.013

• 论著 • 上一篇    下一篇

Ra1362调控环二鸟苷酸影响结核分枝杆菌H37Ra的生物膜发育和休眠

丁晓娟,刘毅(),孙涛,周小丹,李传友,方海红()   

  1. 230032 合肥,安徽医科大学基础医学院微生物学教研室(丁晓娟、孙涛、方海红);首都医科大学附属北京胸科医院 北京市结核病胸部肿瘤研究所耐药结核病重点实验室细菌免疫室(刘毅、李传友);中国科学技术大学生命科学院(周小丹);北京中学生物组(方海红)
  • 收稿日期:2017-12-27 出版日期:2018-06-20 发布日期:2018-07-24
  • 通信作者: 丁晓娟 E-mail:liuyilotus@hotmail.com;fhaihong@aliyun.com
  • 基金资助:
    安徽省高校自然科学研究重点项目(KJ2015A019)

Ra1362 modulates biofilm development and dormancy of Mycobacterium tuberculosis through c-di-GMP in H37Ra

Xiao-juan DING,Yi LIU(),Tao SUN,Xiao-dan ZHOU,Chuan-you LI,Hai-hong FANG()   

  1. *Department of School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
  • Received:2017-12-27 Online:2018-06-20 Published:2018-07-24
  • Contact: Xiao-juan DING E-mail:liuyilotus@hotmail.com;fhaihong@aliyun.com

摘要: 目的

通过敲除环二鸟苷酸(c-di-GMP)合成酶基因Ra1362探讨该基因在结核分枝杆菌(MTB) H37Ra株生物膜发育和休眠中的作用。

方法

以MTB H37Ra株为出发菌株,敲除合成酶基因Ra1362获得基因敲除株△Ra1362,通过体外实验比较细菌的生物膜形成变化;应用转录谱基因芯片和实时荧光定量PCR(RT-PCR)检测野生株和敲除株基因表达的情况变化;同时构建体外快速厌氧休眠模型比较细菌克隆形成、休眠相关基因的表达和活细胞染色情况。

结果

△Ra1362株于痰液管中培养26d时已形成较厚的皱褶生物膜,形成生物膜的速度明显快于野生株5d;转录谱基因芯片和定量RT-PCR结果也显示△Ra1362株中19个与休眠相关基因的表达水平下调并能通过基因回补和外源添加c-di-GMP所补偿;在快速厌氧模型中发现,迟缓期过后△Ra1362株对氧气的消耗比野生株组要快12h,且细菌处于不能恢复正常生长的休眠状态。

结论

Ra1362基因通过控制c-di-GMP的合成调控MTB感应缺氧或者氧化还原压力,一方面调控生物膜的发育,另一方面在缺氧条件下通过调控DosR调节子基因的表达来调节细菌的休眠。

关键词: 分枝杆菌, 结核, 二鸟苷酸环化酶, 基因敲除技术, 生物膜, 基因表达调控

Abstract: Objective

The aim of this study was to explore the modulation of Ra1362, the encoding gene of cyclic guanylic acid (c-di-GMP) synthesis, during the biofilm development and dormancy of Mycobacterium tuberculosis (MTB) in H37Ra by knocking out Ra1362.

Methods

MTB H37Ra strain was selected as the original strain, and the Ra1362 knock out strain (△Ra1362) was constructed. Change in biofilm formation was compared in vitro; gene expression differences between the wild type (WT) strain and △Ra1362 were detected by transcriptional microarray and real-time fluorescent quantitative PCR experiments. Also, rapid anaerobic dormancy model (RADM) was established in vitro to compare the formation of clones, expression of dormancy-related genes and result of live-cell staining.

Results

△ Ra1362 strian had formed a thicker biofilm in the sputum tube on the 26th day of culture, and the formation speed was 5 days faster than that of WT strain. The results of transcriptional microarray and real-time fluorescent quantitative PCR showed that the expression levels of 19 dormancy-related genes were downregulated in △Ra1362, which could be recovered in Ra1362 complementary strain or after adding exogenous c-di-GMP. In the RADM, it was found that the oxygen consumption in △Ra1362 was 12 h faster than that in WT after the lag phase, and the strians were in a dormant state in which normal growth could not be restored.

Conclusion

Ra1362 can modulate the biofilm development and dormancy of MTB H37Ra in vitro, which is based on the gene expression of DosR regulons under hypoxic condition by sensing hypoxia or oxidation-reduction pressure through controling c-di-GMP synthesis.

Key words: Mycobacterium tuberculosis, Di-guanylate cyclase, Gene knockout techniques, Biofilms, Gene expression regulation