Sigma E基因对耻垢分枝杆菌五种药物敏感性的影响及机制研究

目的探讨Sigma E (sigma factor E,SigE)基因影响耻垢分枝杆菌对5种药物的敏感性及其作用机制研究。方法 PCR法扩增出耻垢分枝杆菌sigE基因,克隆入质粒pMV261构建重组pMV261-SigE质粒,测序验证。重组质粒电转入耻垢分枝杆菌,Western blot检测SigE的表达。设立含空质粒转化菌为对照组,采用刃天青作为指示剂的微量滴定板法和菌落计数法检测pMV261-SigE重组耻垢分枝杆菌对异烟肼、利福平、乙胺丁醇、左氧氟沙星和环丙沙星5种抗结核药物的敏感性。采用相应抗生素处理重组耻垢分枝杆菌48 h诱导细菌进入非复制持留状态,随后在含20 ng/mL的结核分枝杆菌复苏促进因子E (resuscitation-promoting factor E, RpfE)的7H9中静置培养2周,计数最大可能数和细菌生长数,测算复苏指数来了解SigE对药物敏感性的影响机制。结果成功构建pMV261-SigE,经测序鉴定序列正确,Western blot检测到SigE在耻垢分枝杆菌中表达。与对照组相比,表达SigE重组耻垢分枝杆菌组对异烟肼、利福平等5种药物的相对荧光百分比高,敏感性下降。5组抗生素中,异烟肼、乙胺丁醇两种药物作用后pMV261-SigE重组耻垢分枝杆菌复苏指数明显高于对照组。结论 SigE可通过促进耻垢分枝杆菌进入持留状态影响异烟肼和乙胺丁醇作用的敏感性。

Effect and mechanism of Sigma E on drug sensitivity of Mycobacterium smegmatis to five antibiotics

The purpose of this study was to explore the effect of SigE on drug sensitivity of Mycobacterium smegmatis to five antibiotics (Isoniazid, Rifampin, Ethambutol, Levofloxacin, and Ciprofloxacin) and its' mechanism. sigE gene was amplified and cloned into plasmid pMV261 to construct recombinant plasmid pMV261-SigE. The pMV261-SigE was transferred into M. smegmatis to construct recombinant M. smegmatis expressing SigE (pMV261-SigE/MS). Expression of sigE gene was detected by Western blot. M. smegmatis carrying only plasmid pMV261 (pMV261/MS) was used as control. We found that pMV261-SigE/MS group was more resistance to five antibiotics than control group by Resazurine Microtiter Assay and Colony-Forming Units (CFU). Further, the recombinant bacteria were induced into non-replication persistence phase treated with corresponding higher concentration antibiotic for 48 hours. Resuscitation-promoting factor E (RpfE) of M. tuberculosis was cloned and expressed to enhance resuscitation and growth of persistence phase bacteria. The bacteria in persistence phase were incubated in 7H9 containing 20 ng/mL of RpfE for 2 weeks. The bacterial CFU and most probable number (MPN) were measured to count resuscitation index (RI). RI was used to assess bacterial persistence ability which help us to understand the mechanism of SigE effecting drug sensitivity. Among five antibiotics, RI of pMV261-SigE/MS groups in Isoniazid and Ethambutol were significantly higher than that of the control group. So, SigE can influence the sensitivity of isoniazid and ethambutol by promoting M. smegmatis into the non-replication persistence phase.