耻垢分枝杆菌新型毒素-抗毒素系统MSMEG_3435-3436基因功能的初步研究

Preliminary Study on the Gene Function of a Novel Toxin-antitoxin System MSMEG_3435-3436 in Mycobacterium smegmatis

目的初步探讨耻垢分枝杆菌(Mycobacterium smegmatis)毒素-抗毒素(toxin-antitoxin,TA)系统基因的功能及其在细菌药物耐受中的作用。方法利用无水四环素(ATe)诱导穿梭质粒构建毒素基因(MSMEG_3436和MSMEG_6760)表达系统,检测毒素基因表达的抑菌作用。应用CRISPR-Cas12a基因编辑技术构建△MSMEG_3435-3436和△MSMEG_6762-6760敲除菌株,探究毒素-抗毒素系统对菌株生长的影响。通过计算菌株存活率检测MSMEG_3435-3436基因对异烟腓(96 ug/ml)和利福平(40 pgml)的耐受相关性。在耻垢分枝杆菌中用LacZ报告基因分别替换毒素-抗毒素基因(MSMEG_1277-1278、MSMEG_1283-1284、MSMEG_3435-3436、MSMEG_4447-448和MSMBG_5635-5634),构建5个启动子活性检测突变菌株(SY3328、SY3309、SY6407、SY310和SY3311),并将pMV261空载体和pMV261-抗毒素系列质粒分别电转至5个突变菌株中,通过测定吸光度值(Aaw.Akso和Ana)计算β-半乳糖苷酶活性[酶活性单位为“Miller单位(MU)”],以检测毒素抗毒素系统的启动子活性。结果在耻垢分枝杆菌中,ATc诱导表达毒素基因MSMEG_3436可抑制细菌生长,而同时表达对应的抗毒素基因MSMEG_3435可消除抑制作用;ATe诱导表达毒素基因MSMEG_6760未发现明显的抑菌作用。与野生株相比,AMSMEG_3435-3436和△MSMEG_6762-6760敲除菌株在TH9液体培养基中生长表型无明显差异。野生株和△MSMEG_3435-3436敲除菌株经异烟阱和利福平处理后的存活率[分别为(4.38±1.48)%和(3.49±0.66)%,(0.15±0.04)%和(0.03±0.02)%]显示毒素-抗毒素基因MSMEG_3435-3436与药物耐受性无关(t=0.548,P=0.613;t=2.663,P=0.056)。启动子(SY328、SY3309、SY6407、SY3310和SY3311)在携带pMV261-空载体和pMV261抗毒素表达质粒的报告菌株中的β-半乳糖苷酶活性分别为(376.50±17.13)和(315.50±20.71)、(189.00±12.24)和(160.70±9.89)(1225.20±9.95)和(211.70±2.57)(221.40±12.07)和(186.60±13.17)(179.10±5.87)和(127.70±19.21)MU,差异均无统计学意义(t=2.272,P=0.086;t=1.795,P=0.147;t=1.319,P=0.258;t=1.949,P=0.123;t=-2.562,P=0.063)。结论成功构建了MSMEG_3435-3436和MSMEG_6762-6760在耻垢分枝杆菌中的诱导表达体系及敲除菌株,并发现MSMEG_3435-3436是一个新的有功能的毒素-抗毒素系统,以及这两个毒素-抗毒素系统与菌株生长表型及异烟阱和利福平耐受性无关,最后发现耻垢分枝杆菌中5对毒索-抗毒素系统的抗毒素基因可能在自身启动子调控中不发挥关键作用,可为进一步研究结核分枝杆菌毒素-抗毒素系统的功能提供线索。

Objective To investigate the function of the toxin-antioxin(TA)systems in Mycobacterium smegmatis(M.smegmatis)and its role in bacterial drug tolerancc.Methods Two toxingenes(MSMEG_3436 and MSMEG_6760)were constructed in anhydrous teracyeline(ATo)-induced shutl.plasmid,respectively and then tested for repression of cell growth in M.smegmatis.The AMSMEG_3435-3436 and AMSMEG_6762-6760 mutants were constructed using CRISPR-Cas12a genome editing system to investigate the effect of TA system on cell growth.Wild type M.smegmatis and MSMEG_3435-3436 deletion mutant were treated with isonmiazid(96 μg/ml)and rifampicin(40 μg/ml)and tested for drug tolerance by calculating survival rate.The TAgenes(MSMEG_1277-1278,MSMEG_1283-1284,MSMEG_3435-3436,MSMBG_4447-448,MSMEG_5635-5634)was replaced by LacZ.reporter gene inM.smegmatis,resulting in promoter activity analysis stains SY3328,SY3309,SY6407,SY3310,and SY3311.The empty vector pMV261 and its derivatives expressing antitoxins were transformed nto the aforementioned strains,respectively.The promoter activity of TAgenes was then assessed by measurement of β-galactosidase.Results Highly expression of toxin gene MSMEG_3436 but not MSMEG_6760 in M.smegmatis repressedcll growth,while co-expression of anti-toxingene MSMEG_3435 relieved the repression.The wild-type,AMSMEG_3435-3436 and △MSMEG_6762-6760 mutants showed similar growth phenotype in TH9 liquid medium.Wild-type strain and AMSMEG_3435-3436 mutant treated with isoniazid and rifampicin had similar survival rate((4.38±1.48)%and(3.49±0.66)%,(0.15±0.04)%and(0.03±0.02)%),which suggested that MSMEG_3435-3436 might not play an important role in drug tolerance of M.smegmatis(t=0.548,P=0.613;t2.663,P-0.056.Promoter activity analysis showed that the β-galactosidase activities(376.50±17.13)and(315.50±20.71)Miller units(MU),(189.00±12.24)and(160.70±9.89)MU,(225.20±9.95)and(211.70±2.57)MU,(221.40±12.07)and(186.60±13.17)MU,(179.10±5.87)and(127.70±19.21)MU)in the reporter strains harboring empty vector and antitoxin expressing plasmids were not significantlydifferent(=2.272,P=0.086;1.795,P-0.147;t=1.319,P=0.258;t=1.949,P=0.123;t=2.562,P-0.063).Conclusion The MSMEG_3435-3436 and MSMEG_6762-6760 induced expression systems and knockout strains are constructed in M.smegmatis.Further study shows that MSMEG_3435-3436 is a functional toxin-antitoxin system,but does not affetcellgrowth and might not affct the tolerance to isoniazid and rifampicin inM.smegmatis.Finally,the antitoxins of the known 5'TA systems are found not critical for auto-regulation of theirpromoters activity in M.smegmatis.These findings provide clues for further investigation of the roles of'TA systems in M.tuberculosis.