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

doi:10.19982/j.issn.1000-6621.20220525

中国防痨杂志 ›› 2023, Vol. 45 ›› Issue (4): 391-400.doi: 10.19982/j.issn.1000-6621.20220525

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

段玉衡, 张蓝月, 董静, 史雨婷, 贾红彦, 李自慧, 邢爱英, 杜博平, 孙琦, 潘丽萍, 朱传智(), 张宗德()

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

收稿日期:2023-01-10 出版日期:2023-04-10 发布日期:2023-03-31
通信作者: 朱传智 Email:chuanzhizhu@gmail.com;张宗德 Email:zzd417@163.com
基金资助:
国家自然科学基金(82172279);国家自然科学基金(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)

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

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

Received:2023-01-10 Online:2023-04-10 Published:2023-03-31
Contact: Zhu Chuanzhi Email:chuanzhizhu@gmail.com; Zhang Zongde Email:zzd417@163.com
Supported by:
National Natural Science Foundation of China(82172279);National Natural Science Foundation of China(82070012);Beijing Hospitals Authority Innovation Studio of Young Staff Funding Support(202136);Tongzhou Yunhe Project(2019B0402264)

摘要/Abstract

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1116bp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A₆₀₀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(72h)和小鼠肺组织中(28d),H37Rv感染后的菌落形成单位计数[分别为(41.000±4.583)×10⁴和log₁₀(5.531±0.203)]与ΔfadA3感染[(18.670±1.155)×10⁴和log₁₀(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。

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

Abstract:

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 and ΔfadA3 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 and ΔfadA3 was analyzed by Western blotting and transcriptome sequencing. The survival and pathological modifications of H37Rv and ΔfadA3 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 the ΔfadA3 knockout strain. There was no significant difference in the A₆₀₀ values between H37Rv and ΔfadA3 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 MIC₉₀ of isoniazid, ethambutol, streptomycin, levofloxacin, PA-824, linezolid, clofazimine, rifampicin, bedaquiline, delamanid and other first and second-line antituberculosis drugs on ΔfadA3 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, 0.320 μg/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) and ΔfadA3 infected group (154.500±14.890)(t=5.294, P=0.013; t=9.350, P=0.003). In ΔfadA3 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)×10⁴ and (18.670±1.155)×10⁴, log₁₀ (5.531±0.203) and log₁₀ (4.541±0.276)) after H37Rv and ΔfadA3 infection in the macrophage model (72h) and mouse lung tissue (28d)(t=8.815, P=0.001; t=6.466, P<0.001). The pathological inflammatory infiltration of lung tissue in mice infected with ΔfadA3 was significantly weaker than in H37Rv-infected mice. Conclusion: The germicidal efficacy of anti-tuberculosis drugs on ΔfadA3 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.

Key words: Mycobacterium tuberculosis, Acetyltransferase, Mononuclear phagocyte system, Acetylation, Gene expression, Cell survival

中图分类号:

段玉衡, 张蓝月, 董静, 史雨婷, 贾红彦, 李自慧, 邢爱英, 杜博平, 孙琦, 潘丽萍, 朱传智, 张宗德. 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[J]. 中国防痨杂志, 2023, 45(4): 391-400. doi: 10.19982/j.issn.1000-6621.20220525

Duan Yuheng, Zhang Lanyue, Dong Jing, Shi Yuting, Jia Hongyan, Li Zihui, Xing Aiying, Du Boping, Sun Qi, Pan Liping, Zhu Chuanzhi, Zhang Zongde. Effects of acetyltransferase fadA3 on acetylation of host protein and in vivo survival of Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2023, 45(4): 391-400. doi: 10.19982/j.issn.1000-6621.20220525

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GO地址	聚类名称	富集基因数 (个)	构成比 (%)	Q值
分子功能分析
GO:0005125	细胞因子活性(cytokine activity)	12	5	<0.001
GO:0008009	趋化因子活性(chemokine activity)	6	12	<0.001
GO:0048248	CXCR3趋化因子受体结合(CXCR3 chemokine receptor binding)	3	60	<0.001
GO:0045236	CXCR趋化因子受体结合(CXCR chemokine receptor binding)	3	20	0.004
GO:0008201	肝素结合(heparin binding)	7	4	0.004
GO:0004992	血小板活化因子受体活性(platelet activating factor receptor activity)	2	50	0.008
GO:0031727	CCR2趋化因子受体结合(CCR2 chemokine receptor binding)	2	33	0.017
GO:0048020	CCR趋化因子受体结合(CCR chemokine receptor binding)	3	10	0.020
生物进程分析
GO:0060337	Ⅰ型干扰素信号通路(type Ⅰ interferon signaling pathway)	11	16	<0.001
GO:0006955	免疫应答(immune response)	18	5	<0.001
GO:0002376	免疫系统进程(immune system process)	20	4	<0.001
GO:0045071	病毒基因组复制的负调控(negative regulation of viral genome replication)	8	18	<0.001
GO:0045087	先天免疫反应(innate immune response)	18	4	<0.001
GO:0070098	细胞因子介导信号通路(cytokine-mediated signaling pathway)	8	11	<0.001
GO:0006935	趋化作用(chemotaxis)	10	6	<0.001
GO:0006954	炎症应答(inflammatory response)	13	3	<0.001
GO:0009617	细菌反应(response to bacterium)	7	6	<0.001
GO:0030593	中性粒细胞趋化性(neutrophil chemotaxis)	6	7	<0.001
细胞组分分析
GO:0005576	胞外区域(extracellular region)	29	1	0.001
GO:0005634	细胞外隙(extracellular space)	22	1	0.002

通路编码	通路名称	富集基因数 (个)	构成比 (%)	Q值
hsa04061	病毒蛋白与细胞因子和细胞因子受体的相互作用(viral protein interaction with cytokine and cytokine receptor)	10	10	<0.001
hsa04060	细胞因子-细胞因子受体相互作用(cytokine-cytokine receptor interaction)	15	5	<0.001
hsa05160	丙型病毒性肝炎(hepatitis C)	8	5	0.001
hsa05164	甲型流感病毒(influenza A)	8	5	0.002
hsa04062	趋化因子信号通路(chemokine signaling pathway)	8	4	0.003
hsa04620	Toll样受体信号通路(Toll-like receptor signaling pathway)	5	5	0.027
hsa04623	胞质DNA传感途径(cytosolic DNA-sensing pathway)	4	6	0.028
hsa04622	RIG-Ⅰ样受体信号通路(RIG-Ⅰ-like receptor signaling pathway)	4	6	0.036
hsa04621	NOD样受体信号通路(NOD-like receptor signaling pathway)	6	3	0.042

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

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时间(d)	H37Rv	ΔfadA3	t值	P值
0	0.065±0.006	0.058±0.001	1.971	0.120
3	0.245±0.005	0.232±0.013	1.623	0.180
6	0.403±0.122	0.385±0.009	2.351	0.057
9	0.444±0.010	0.442±0.005	0.178	0.866
12	0.675±0.027	0.662±0.026	0.848	0.416

组别	灰度值	t值	P值^a	t值	P值^b	t值	P值^c
未感染组	204.800±9.348	-	-	5.294	0.013	4.042	0.056
H37Rv感染组	243.100±7.125	5.294	0.013	-	-	9.350	0.003
ΔfadA3感染组	154.500±14.890	4.042	0.056	9.350	0.003	-	-

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