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

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)]的差异均有统计学意义(t=8.815,P=0.001;t=6.466,P<0.001);ΔfadA3感染小鼠肺组织病理炎症性浸润较H37Rv感染小鼠明显减弱。结论:抗结核药物对ΔfadA3敲除株与H37Rv的杀菌效果一致。fadA3可显著调控宿主蛋白乙酰化修饰和基因表达,可能是维持H37Rv在巨噬细胞胞内和小鼠肺组织中存活,并导致肺组织炎症性浸润的重要毒力因子。这些发现将为靶向fadA3和宿主蛋白乙酰化的宿主导向治疗提供理论数据。

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 up-regulated,and 7 were down-regulated compared to H37Rv infection.Moreover,there were significant differences in intracellular CFU count((41.000±4.583)×10^(4)and(18.670±1.155)×10^(4),log 10(5.531±0.203)and log 10(4.541±0.276))after H37Rv andΔfadA3 infection in the macrophage model(72 h)and mouse lung tissue(28 d)(t=8.815,P=0.001;t=6.466,P<0.001).The pathological inflammatory infiltration of lung tissue in mice infected withΔfadA3 was significantly weaker than in H37Rv-infected mice.Conclusion:The germicidal efficacy of anti-tuberculosis drugs onΔfadA3 knockout strain was equivalent to H37Rv.The acetylation modification of host protein and gene expression could be significantly regulated by fadA3,which may act as an essential virulence factor for maintaining the survival of H37Rv in macrophages and mouse lung tissue,leading to the inflammatory infiltration of lung tissue.These finding provides the theoretical data for host-directed therapy targeting fadA3 and host protein acetylation.