结核分枝杆菌乙酰转移酶fadA3对宿主蛋白乙酰化修饰及其体内存活影响的研究  

Effects of acetyltransferase fadA3 on acetylation of host protein and in vivo survival of Mycobacterium tuberculosis

作　　者：段玉衡 张蓝月 董静 史雨婷 贾红彦[1] 李自慧[1] 邢爱英[1] 杜博平 孙琦[1] 潘丽萍[1] 朱传智 张宗德[1] Duan Yuheng;Zhang Lanyue;Dong Jing;Shi Yuting;Jia Hongyan;Li Zihui;Xing Aiying;Du Boping;Sun Qi;Pan Liping;Zhu Chuanzhi;Zhang Zongde(Laboratory of Molecular Biology,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/分子生物学实验室,北京101149

出　　处：《中国防痨杂志》2023年第4期391-400,共10页Chinese Journal of Antituberculosis

基　　金：国家自然科学基金(82172279;82070012);北京市医院管理中心青年职工创新工作室-创新梦工场(202136);通州区运河人才计划(2019B0402264)。

摘　　要：目的:探讨结核分枝杆菌(Mycobacterium tuberculosis,MTB)乙酰转移酶fadA3对宿主蛋白乙酰化修饰、基因表达和MTB在体内存活的影响。方法:从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分子生物学实验室选取MTB标准株(H37Rv),利用CRISPR-cas系统辅助的非同源性末端接合技术(CRISPR-NHEJ)构建H37Rv-fadA3基因敲除株(ΔfadA3),利用微孔板法分别检测H37Rv和ΔfadA3在7H9液体培养基中的吸光度值(A值)和细菌活性,绘制生长曲线图和最低抑菌浓度表。运用免疫印迹和转录组学测序分析H37Rv和ΔfadA3感染巨噬细胞(巨噬细胞系THP-1分化得到)后蛋白质乙酰化修饰变化及基因表达的差异情况;采用菌落计数和苏木精-伊红染色法分析H37Rv和ΔfadA3在C57BL/6J小鼠肺组织和巨噬细胞中的存活及病理变化。结果:fadA3经敲除1116 bp片段后成功获得ΔfadA3敲除株。H37Rv和ΔfadA3在培养第3、6、9和12天的A 600值(分别为0.245±0.005和0.232±0.013、0.403±0.122和0.385±0.009、0.444±0.010和0.442±0.005、0.675±0.027和0.662±0.026)差异均无统计学意义(t值分别为1.623、2.351、0.178、0.848,P值均>0.05);二者对异烟肼、乙胺丁醇、链霉素、左氧氟沙星、PA-824、利奈唑胺、氯法齐明、利福平、贝达喹啉、德拉马尼等一线/二线抗结核药物的90%最低抑菌浓度相同(分别为0.006、2.000、0.313、0.156、0.250、0.625、1.250、0.003、0.125、0.320μg/ml)。H37Rv感染巨噬细胞中全蛋白乙酰化修饰灰度值(243.100±7.125)与未感染组(204.800±9.348)和ΔfadA3感染组(154.500±14.890)的差异均有统计学意义(t=5.294,P=0.013;t=9.350,P=0.003)。与H37Rv相比,ΔfadA3感染上调的差异基因有94个,下调有7个。同时,在巨噬细胞模型(72 h)和小鼠肺组织中(28 d),H37Rv感染后的菌落形成单位计数[分别为(41.000±4.583)×10^(4)和log 10(5.531±0.203)]与ΔfadA3感染[(18.670±1.155)×10^(4)和log 10(4.541±0.276)]的差异均有�Objective:To investigate the effects of acetyltransferase fadA3 in Mycobacterium tuberculosis(MTB)on acetylation modification of host protein,gene expression,and MTB survival in vivo.Methods:MTB standard strain(H37Rv)was selected from the Molecular Biology Laboratory of Institute of Tuberculosis and Thoracic Tumor/Beijing Chest Hospital,and the H37Rv-fadA3 knockout gene strain(ΔfadA3)was constructed by CRISPR-cas system-assisted non-homologous terminal conjugation(CRISPR-NHEJ).The absorbance(A value)and bacterial activity of H37Rv andΔfadA3 were detected by microplate method in 7H9 liquid medium,and the growth curve and minimum inhibitory concentration(MIC)table were drawn.The changes in protein acetylation and gene expression of macrophages infected with H37Rv andΔfadA3 was analyzed by Western blotting and transcriptome sequencing.The survival and pathological modifications of H37Rv andΔfadA3 in lung tissue and macrophages of C57BL/6J mice was analyzed by colony count and hematoxylin-eosin staining.Results:The 1116 bp fragment of fadA3 was successfully knocked out to obtain theΔfadA3 knockout strain.There was no significant difference in the A 600 values between H37Rv andΔfadA3 on days 3,6,9,and 12 of cultivation(0.245±0.005 and 0.232±0.013,0.403±0.122 and 0.385±0.009,0.444±0.010 and 0.442±0.005,0.675±0.027 and 0.662±0.026,respectively)(t values were 1.623,2.351,0.178,0.848,respectively,all Ps>0.05).The MIC 90 of isoniazid,ethambutol,streptomycin,levofloxacin,PA-824,linezolid,clofazimine,rifampicin,bedaquiline,delamanid and other first and second-line antituberculosis drugs onΔfadA3 was the same as that on H37Rv(0.006,2.000,0.313,0.156,0.250,0.625,1.250,0.003,0.125,0.320μg/ml,respectively).The grey value of acetylation modification in macrophages whole protein infected with H37Rv(243.100±7.125)was significantly different from that in the uninfected group(204.800±9.348)andΔfadA3 infected group(154.500±14.890)(t=5.294,P=0.013;t=9.350,P=0.003).InΔfadA3 infection group,94 differential genes were

关 键 词：分枝杆菌 结核 乙酰转移酶 单核巨噬细胞系统 乙酰化作用 基因表达 细胞存活 

分 类 号：R378.91[医药卫生—病原生物学] Q344.13[医药卫生—基础医学]