PhoPR双组分信号转导系统对结核分枝杆菌持留性的调控机制研究

Regulatory mechanisms of PhoPR two-component signal transduction system in Mycobacterium tuberculosis persistence

目的本研究通过诱导结核分枝杆菌在低氧环境中进入持留状态,来比较分析不同时间不同低氧环境下结核分枝杆菌中的PhoP基因和PhoR基因的表达水平差异,探讨研究PhoPR双组分信号转导系统对结核分枝杆菌持留性的调控机制。方法首先对结核分枝杆菌国际标准无毒株(H37Ra)和结核分枝杆菌国际标准强毒株(H37Rv)在不同低氧环境下进行培养,提取每个样本菌株的总RNA,并进行完整性鉴定;运用SYBR Green I实时荧光定量PCR检测每个样本菌株中PhoP基因和PhoR基因的表达水平;比较分析不同时间不同低氧环境下的结核分枝杆菌菌株PhoP基因和PhoR基因的表达水平差异。结果在不同时间点对低氧环境的结核分枝杆菌PhoP基因和PhoR基因的表达水平进行检测,结果显示:与第10d相比,培养至第15d时H37Rv菌株和H37Ra菌株中的PhoP基因和PhoR基因的表达水平均显著上调,差异有统计学意义(P<0.05);并且培养至第25d时,H37Rv菌株中的PhoP基因和PhoR基因的表达水平比H37Ra菌株表达均上调2.34倍,差异有统计学意义(P<0.05)。结论在不同时间点相同毒力的结核分枝杆菌的PhoPR双组分信号转导系统中PhoP基因和PhoR基因在不同低氧环境下的表达存在差异,而且在相同时间点不同毒力结核分枝杆菌的PhoPR双组分信号转导系统中PhoP基因和PhoR基因在不同低氧环境下的表达也存在差异,表明PhoPR双组分信号转导系统对结核分枝杆菌的持留性具有调控作用。

In this study,we explored the regulatory mechanisms of PhoPR two-component signal transduction system in Mycobacterium tuberculosis persistence by analyzing the expression levels difference of PhoP gene and PhoR gene at various time in different oxygen conditions of Mycobacterium tuberculosis which were induced persistent state under low-oxygen conditions.The international standard avirulent strains of Mycobacterium tuberculosis（H37Ra）and the international standard virulent strains of Mycobacterium tuberculosis（H37Rv）were cultured under different low-oxygen conditions,then the total RNA extracted from each sample strain and the integrity of the RNA were checked by gel electrophoresis.The expression of PhoP gene and PhoR gene were quantified by using SYBR Green I FQ-PCR.The expression levels difference of these genes were compared in different low-oxygen conditions of Mycobacterium tuberculosis.The expression levels of PhoP gene and PhoR gene of Mycobacterium tuberculosis under low-oxygen conditions were measured at various times.The expression level of PhoP gene and PhoR gene of H37 Rv strain and H37 Ra strain cultured for 15 days compared with 10 days were significantly up-regulated（P〈0.05）;and the expression level of PhoP gene and PhoR gene of H37 Rv strain cultured for 25 days were up-regulated by 2.34 times compared with H37 Ra strain（P〈0.05）.In this study,there are differences under different low-oxygen conditions in the expression levels of PhoP gene and PhoR gene of Mycobacterium tuberculosis PhoPR two-component signal transduction system at various time and same virulent strains,and there are differences under the same low-oxygen condition in the expression levels of PhoP gene and PhoR gene of Mycobacterium tuberculosis PhoPR two-component signal transduction system at the same time and different virulent strains.Therefore,the system plays a regulatory role in Mycobacterium tuberculosis persistence.