硫醇乙酰基转移酶mshD对结核分枝杆菌生长和应对压力刺激的研究  

Study on the effect of thiol acetyltransferase mshD on the growth and stress response of Mycobacterium tuberculosis

作　　者：张蓝月 王颖超 刘唯夷 尚雪恬 贾红彦[1] 朱传智 张宗德[1] 潘丽萍[1] Zhang Lanyue;Wang Yingchao;Liu Weiyi;Shang Xuetian;Jia Hongyan;Zhu Chuanzhi;Zhang Zongde;Pan Liping(Beijing Key Laboratory of Drug-resistant Tuberculosis Research,Institute of Tuberculosis and Thoracic Tumor,Beijing Chest Hospital,Capital Medical University,Beijing 101149,China)

机构地区：[1]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/耐药结核病研究北京市重点实验室,北京101149

出　　处：《中国防痨杂志》2024年第8期935-941,共7页Chinese Journal of Antituberculosis

基　　金：通州区卫生发展科研储备项目(KJ2024CX064);北京市自然科学基金(7242025);北京市医院管理中心青年职工创新工作室(202136)。

摘　　要：目的:探索硫醇乙酰基转移酶MshD对结核分枝杆菌在体外生长、应对压力刺激和活性氧(reactive oxygen species,ROS)水平的影响。方法:利用CRISPR-NHEJ基因编辑技术,构建结核分枝杆菌mshD基因敲除株(ΔmshD株)和回补株(ΔmshD::mshD株)。分别检测H37Rv野生株(WT)、ΔmshD株和ΔmshD::mshD株在液体培养基和固体培养基中的生长情况,以及外源添加L-半胱氨酸和过氧化氢酶对菌株生长的影响;检测3种菌株对不同刺激剂如H 2O 2、二硫苏糖醇(DTT)、十二烷基硫酸钠(SDS)的敏感性,以及外源添加过氧化氢酶对压力条件处理后菌株的恢复情况;采用流式细胞术检测SDS处理3种菌株前后菌株的ROS水平。结果:与WT和ΔmshD::mshD株相比,ΔmshD株在固体平板上的生长较为缓慢,外源添加过氧化氢酶可以恢复其生长趋势;ΔmshD株应对DTT[WT(6.96±2.02)%,ΔmshD(0.02±0.00)%,ΔmshD::mshD(6.64±0.77)%;F=29.700,P<0.001],H 2O 2[WT(0.23±0.06)%,ΔmshD(0.01±0.00)%,ΔmshD::mshD(0.26±0.06)%;F=25.520,P=0.001]和SDS[WT(0.12±0.03)%,ΔmshD(0.01±0.00)%,ΔmshD::mshD(0.18±0.04)%;F=19.540,P=0.002]刺激的存活率更低,差异均有统计学意义;外源添加过氧化氢酶可以恢复其存活率。ΔmshD株自身ROS水平高于WT(WT:95.100±2.553,ΔmshD:106.000±4.000,ΔmshD::mshD:94.667±3.055;F=11.650,P=0.009),SDS处理ΔmshD株的自身ROS水平进一步升高(WT:436.000±8.000,ΔmshD:533.667±4.726,ΔmshD::mshD:441.333±2.517;F=292.900,P<0.001),差异均有统计学意义。结论:mshD基因缺失在固体培养基中生长减慢,mshD基因协助结核分枝杆菌抵抗各种应激压力,ΔmshD菌株内源性ROS水平增加。外源添加过氧化氢酶可一定程度恢复mshD基因敲除株生长和存活缺陷。Objective:To investigate the influence of thiol acetyltransferase MshD on the growth dynamics,stress resilience,and reactive oxygen species(ROS)modulation in Mycobacterium tuberculosis(MTB)under in vitro conditions.Methods:Employing CRISPR-NHEJ gene editing,this study established an mshD gene knockout strain(ΔmshD)and a complemented strain(ΔmshD::mshD).We monitored the growth trajectories of the H37Rv wild-type strain(WT),theΔmshD strain,and theΔmshD::mshD strain in both liquid and solid media.Additionally,we assessed the impact of exogenously added L-cysteine and catalase on the growth of these strains.The sensitivity of the strains to various stressors,including H 2O 2,dithiothreitol(DTT),and sodium dodecyl sulfate(SDS),and their recovery post-stress intervention with exogenous catalase were evaluated.Furthermore,flow cytometry was utilized to measure the ROS levels in the strains both prior to and following SDS exposure.Results:TheΔmshD strain exhibited significantly slower growth on solid media compared to the WT andΔmshD::mshD strains.However,the introduction of exogenous catalase reinstated their growth patterns to near-normal levels.In survival assays,theΔmshD strain showed markedly reduced resilience against DTT(WT:(6.96±2.02)%,ΔmshD:(0.02±0.00)%,ΔmshD::mshD:(6.64±0.77)%;F=29.700,P<0.001),H 2O 2(WT:(0.23±0.06)%,ΔmshD:(0.01±0.00)%,ΔmshD::mshD:(0.26±0.06)%;F=25.520,P=0.001),and SDS(WT:(0.12±0.03)%,ΔmshD:(0.01±0.00)%,ΔmshD::mshD:(0.18±0.04)%;F=19.540,P=0.002),with all differences reaching statistical significance.Catalase supplementation notably restored the survival rate of theΔmshD strain.Additionally,ROS levels in theΔmshD strain were elevated compared to WT(WT:95.100±2.553,ΔmshD:106.000±4.000,ΔmshD::mshD:94.667±3.055;F=11.650,P=0.009)and further increased following SDS exposure(WT:436.000±8.000,ΔmshD:533.667±4.726,ΔmshD::mshD:441.333±2.517;F=292.900,P<0.001),underlining significant oxidative stress responses.Conclusion:Deletion of the mshD gene impairs growth in solid cu

关 键 词：分枝杆菌 结核 乙酰基转移酶类 过氧化氢酶 活性氧 

分 类 号：R52[医药卫生—内科学] R378.91[医药卫生—临床医学]