【摘要】 目的 探究Rv0559c和Rv0560c基因在结核分枝杆菌外排利福平中的作用，并初步探讨其分子机制。方法 25株利福平耐药结核分枝杆菌临床分离株和6株对4种一线抗结核药物敏感的结核分枝杆菌临床分离株均来自广州市胸科医院（2018-2022年），采用微孔板阿尔玛蓝法（MABA）检测不含或含80μg/mL外排泵抑制剂维拉帕米时25株临床分离株对利福平的最小抑菌浓度（MIC）；实时荧光定量PCR(RT-qPCR)检测利福平敏感菌株和耐药菌株暴露于0.5倍MIC浓度利福平培养10 d的Rv0559c和Rv0560c基因转录水平，以及利福平耐药菌株暴露于0.5倍MIC浓度利福平和0.5倍MIC浓度利福平+80μg/mL维拉帕米培养10 d的Rv0559c和Rv0560c基因表达水平；使用pyrx软件进行分子对接预测Rv2887与利福平和维拉帕米的结合能。结果 在维拉帕米作用下，利福平耐药菌株对利福平的MIC下降4～32倍；将利福平耐药菌株分为耐多药（MDR）菌株（n=12）和非MDR菌株（n=13），在维拉帕米作用下，MDR菌株和非MDR菌株分别有8.3%和30.8%菌株对利福平的MIC下降32倍。与利福平敏感菌株比较，利福平耐药菌株在利福平作用后，Rv0559c和Rv0560c基因表达显著上调，差异均有统计学意义（t=-4.512、-6.491,P均<0.001）；与0.5倍MIC浓度利福平作用比较，利福平耐药菌株在0.5倍MIC浓度利福平+80μg/mL维拉帕米作用下，Rv0559c和Rv0560c基因表达水平出现显著下调，差异均有统计学意义（t=5.339、6.208,P均<0.001）。分子对接显示，利福平和维拉帕米与Rv2887的结合能评分为-7.0和-5.1 Kcal/mol，与Rv2887的结合均具有良好稳定性。结论 利福平与Rv2887的结合可能正反馈调控Rv0559c和Rv0560c基因的表达进而加强结核分枝杆菌外排利福平的作用。更多还原

【Abstract】 Objective To investigate the role of Rv0559c and Rv0560c genes in rifampicin efflux by Mycobacterium tuberculosis and to preliminarily explore their molecular mechanisms. Methods Twenty-five rifampicin-resistant Mycobacterium tuberculosis clinical isolates and six Mycobacterium tuberculosis clinical isolates susceptible to four first-line anti-tuberculosis drugs were collected from Guangzhou Chest Hospital between 2018 and 2022. The minimum inhibitory concentration(MIC) of rifampicin in the absence or presence of the efflux pump inhibitor verapamil(80 μg/mL) was determined using microtiter plate alamar blue assay(MABA). Real-time quantitative PCR(RT-qPCR) was used to detect the transcriptional levels of Rv0559c and Rv0560c genes in rifampicin-susceptible and resistant strains exposed to 0.5 fold MIC rifampicin for 10 days, as well as in rifampicin-resistant strains exposed to 0.5 fold MIC rifampicin and 0.5 fold MIC rifampicin+80 μg/mL verapamil for 10 days. Molecular docking was performed using pyrx software to predict the binding energies of Rv2887 with rifampicin and verapamil. Results The MICs of rifampicin-resistant strains to rifampicin decreased 4-to 32-fold in the presence of verapamil. Rifampicin-resistant strains were divided into multi-drug resistant(MDR) strains(n=12) and non-MDR strains(n=13). Verapamil treatment revealed a 32-fold decrease in the MICs of 8.3% of MDR strains and 30.8% of non-MDR strains to rifampicin. Compared with rifampicin-susceptible strains, rifampicin-resistant strains showed significant upregulation of Rv0559c and Rv0560c gene expression after treatment with rifampicin(t=-4.512,-6.491;both P<0.001). Compared with treatment with 0.5 fold MIC rifampicin, rifampicin-resistant strains showed significant downregulation of Rv0559c and Rv0560c gene expression after treatment with 0.5 fold MIC rifampicin+80 μg/mL verapamil(t=5.339, 6.208;both P<0.001). Molecular docking revealed that the binding energy scores of rifampicin and verapamil with Rv2887 were-7.0 and-5.1 kcal/mol, respectively, indicating good binding stability. Conclusion The binding of rifampicin to Rv2887 might positively feedback regulate the expression of Rv0559c and Rv0560c genes, thereby enhancing the efflux of rifampicin by Mycobacterium tuberculosis.更多还原