pks5基因(Rv1527c)缺失削弱结核分枝杆菌巨噬细胞内生存能力并调控炎症因子表达

doi:10.19983/j.issn.2096-8493.20250101

结核与肺部疾病杂志 ›› 2025, Vol. 6 ›› Issue (6): 747-752.doi: 10.19983/j.issn.2096-8493.20250101

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pks5基因(Rv1527c)缺失削弱结核分枝杆菌巨噬细胞内生存能力并调控炎症因子表达

刘权贤¹, 赵昭亮², 宗兆婧¹, 陈玲¹, 兰远波¹()

¹遵义医科大学附属医院结核病科,遵义 563000
²贵州航天医院呼吸与危重病医学科,遵义 563000

收稿日期:2025-07-16 出版日期:2025-12-20 发布日期:2025-12-08
通信作者: 兰远波,Email:lybivy@163.com
作者简介:注:赵昭亮和刘权贤对本文有同等贡献,为并列第一作者
基金资助:
贵州省卫生健康委科学技术基金项目(gzwjkj2019-1-085);贵州省卫生健康委科学技术基金项目(gzwkj2024-335);贵州省基础研究计划重点项目(黔科合基础-ZK〔2023〕重点060);遵义市科技局科学技术基金项目(遵义科合HZ字(2020)255号)

Deletion of the pks5 gene (Rv1527c) impairs intracellular survival in macrophages and modulates inflammatory cytokine expression in Mycobacterium tuberculosis

Liu Quanxian¹, Zhao Zhaoliang², Zong Zhaojing¹, Chen Ling¹, Lan Yuanbo¹()

¹Tuberculosis Ward, Department of Respiratory and Critical Care Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi 563000, China
²Department of Respiratory Medicine, Guizhou Aerospace Hospital, Zunyi 563000, China

Received:2025-07-16 Online:2025-12-20 Published:2025-12-08
Contact: Lan Yuanbo, Email: lybivy@163.com
Supported by:
Guizhou Provincial Health Commission Science and Technology Fund Project(gzwjkj2019-1-085);Guizhou Provincial Health Commission Science and Technology Fund Project(gzwkj2024-335);Guizhou Provincial Basic Research Program Key Project (QKHJC-ZK〔2023〕ZD060);Zunyi Science and Technology Bureau Fund Project (ZYKJHZ(2020)255)

摘要/Abstract

摘要：

目的: 探究pks5(Rv1527c)基因在结核分枝杆菌致病机制中的作用。方法: 利用噬菌体介导的同源重组技术构建结核分枝杆菌标准株H37RvΔRv1527c基因敲除株;通过抗酸杆菌染色和固体培养基生长曲线,分析细菌形态与生长特性;建立RAW264.7巨噬细胞感染模型,检测细菌胞内存活率;采用实时荧光定量PCR(qPCR)检测感染巨噬细胞中炎症因子[白细胞介素(IL)-1β,肿瘤坏死因子(TNF)-α,IL-6]的mRNA表达水平。结果: pks5敲除株(H37RvΔRv1527c)与野生型H37Rv(H37Rv-WT)菌株在形态上无明显差异。感染72h后,H37RvΔRv1527c在RAW264.7巨噬细胞内的存活率(13.13%±1.05%)较H37Rv-WT的存活率(41.04%±2.11%)明显降低68%(t=15.732,P<0.001)。此外,与感染H37Rv-WT菌株相比,H37RvΔRv1527c感染后巨噬细胞IL-1βmRNA表达量在感染2h即明显上调(H37Rv-WT:1.05±0.08;H37RvΔRv1527c:1.52±0.11;t=3.245,P=0.023),并在感染4h、8h及12h持续维持于更高水平;TNF-α mRNA表达量亦在感染后4h(H37Rv-WT:1.12±0.10;H37RvΔRv1527c:1.98±0.15;t=4.562,P=0.010)与12h(H37Rv-WT:2.05±0.16;H37RvΔRv1527c:3.12±0.21;t=5.328,P=0.006)均出现明显升高。而对于IL-6,在感染24h后,H37RvΔRv1527c感染组的表达水平(1.42±0.10)较H37Rv-WT感染组(3.15±0.22)被明显抑制(t=11.732,P<0.001)。结论: pks5基因是结核分枝杆菌的一个潜在关键毒力因子,通过影响细菌在巨噬细胞内的存活能力并调控宿主炎症反应(促进 IL-1β/TNF-α表达,抑制 IL-6 表达),从而参与其致病过程。

关键词: 分枝杆菌,结核, 聚酮合成酶类, 巨噬细胞, 白细胞介素类, 肿瘤坏死因子α

Abstract:

Objective: To investigate the role of the pks5 (Rv1527c) gene in the pathogenic mechanism of Mycobacterium tuberculosis. Methods: The H37RvΔRv1527c knockout strain was constructed using phage-mediated homologous recombination. Bacterial morphology and growth characteristics were analyzed via acid-fast staining and solid-medium growth curves. A RAW264.7 macrophage infection model was established to assess bacterial intracellular survival rates. qPCR was employed to detect mRNA expression levels of inflammatory cytokines (IL-1β, TNF-α, IL-6) in infected macrophages. Results: The ΔRv1527c knockout strain showed no significant morphological differences compared to wild-type H37Rv. At 72 hours post-infection, the intracellular survival rate of the ΔRv1527c knockout strain in RAW264.7 macrophages (13.13%±1.05%) was significantly reduced by 68% compared to that of the wild-type strain (41.04%±2.11%)(t=15.732, P<0.001). Furthermore, relative to infection with the wild-type H37Rv strain, macrophages infected with the ΔRv1527c knockout strain exhibited a significant upregulation of IL-1β mRNA expression as early as 2 h (H37Rv-WT:1.05±0.08; H37RvΔRv1527c: 1.52±0.11; t=3.245, P=0.023), which remained at significantly higher levels at 4 h, 8 h, and 12 h. Similarly, TNF-α mRNA expression was significantly elevated at 4 h (H37Rv-WT: 1.12±0.10; H37RvΔRv1527c: 1.98±0.15; t=4.562, P=0.010) and 12 h (H37Rv-WT: 2.05±0.16; H37RvΔRv1527c: 3.12±0.21; t=5.328, P=0.006) post-infection. In contrast, for IL-6, its expression level in the ΔRv1527c knockout strain-infected group (1.42±0.10) was significantly suppressed at 24 h post-infection compared to the wild-type H37Rv-infected group (3.15±0.22)(t=11.732, P<0.001). Conclusion: The pks5 gene is a potential key virulence factor in Mycobacterium tuberculosis. It contributes to pathogenesis by impairing bacterial survival within macrophages and modulating host inflammatory responses (promoting IL-1β/TNF-α expression and suppressing IL-6 expression).

Key words: Mycobacterium tuberculosis, Polyketide synthases, Macrophages, Interleukins, Tumor necrosis factor-alpha

中图分类号:

R378.91

刘权贤, 赵昭亮, 宗兆婧, 陈玲, 兰远波. pks5基因(Rv1527c)缺失削弱结核分枝杆菌巨噬细胞内生存能力并调控炎症因子表达[J]. 结核与肺部疾病杂志, 2025, 6(6): 747-752. doi: 10.19983/j.issn.2096-8493.20250101

Liu Quanxian, Zhao Zhaoliang, Zong Zhaojing, Chen Ling, Lan Yuanbo. Deletion of the pks5 gene (Rv1527c) impairs intracellular survival in macrophages and modulates inflammatory cytokine expression in Mycobacterium tuberculosis[J]. Journal of Tuberculosis and Lung Disease, 2025, 6(6): 747-752. doi: 10.19983/j.issn.2096-8493.20250101

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参考文献 17

[1]	Liu W, Wang Y, Zhao J, et al. Identification of Mycobacterium tuberculosis intracellular survival-related virulence factors via CRISPR-based eukaryotic-like secretory protein mutant library screen. Microbiol Spectr, 2025, 13(8): e0076725. doi:10.1128/spectrum.00767-25.
[2]	Dheda K, Mirzayev F, Cirillo DM, et al. Multidrug-resistant tuberculosis. Nat Rev Dis Primers, 2024, 10(1): 22. doi:10.1038/s41572-024-00504-2.
[3]	张蓝月, 王颖超, 刘唯夷, 等. 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. 中国防痨杂志, 2024, 46(8):935-941. doi:10.19982/j.issn.1000-6621.20240184.
[4]	Sun MR, Xing JY, Li XT, et al. Recent advances in research on Mycobacterium tuberculosis virulence factors and their role in pathogenesis. J Microbiol Immunol Infect, 2025, 58(5): 497-507. doi:10.1016/j.jmii.2025.03.017.
[5]	Zhou Z, Wattiez R, Constant P, et al. Telacebec Interferes with Virulence Lipid Biosynthesis Protein Expression and Sensitizes to Other Antibiotics. Microorganisms, 2023, 11(10): 2469. doi:10.3390/microorganisms11102469.
[6]	Prozorov AA, Fedorova IA, Bekker OB, et al. The virulence factors of Mycobacterium tuberculosis: genetic control, new conceptions. Genetika, 2014, 50(8): 885-908. pmid: 25731019
[7]	Boritsch EC, Frigui W, Cascioferro A, et al. pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence. Nat Microbiol, 2016, 1: 15019. doi:10.1038/nmicrobiol.2015.19. pmid: 27571976
[8]	董子璇, 孙俊杰, 代兴杨, 等. 猪链球菌SSU0468基因缺失株的构建及其对菌株形态和抗氧化能力的影响. 畜牧与兽医, 2025, 57(3): 111-117.
[9]	Liu S, Guan L, Peng C, et al. Mycobacterium tuberculosis suppresses host DNA repair to boost its intracellular survival. Cell Host Microbe, 2023, 31(11): 1820-1836. e10. doi:10.1016/j.chom.2023.09.010.
[10]	Xander C, Rajagopalan S, Jacobs WR Jr, et al. The SapM phosphatase can arrest phagosome maturation in an ESX-1 independent manner in Mycobacterium tuberculosis and BCG. Infect Immun, 2024, 92(7): e0021724. doi:10.1128/iai.00217-24.
[11]	Madduri BTSA, Bell SL. Bug in the code: TB blocks DNA repair. Cell Host Microbe, 2023, 31(11): 1769-1771. doi:10.1016/j.chom.2023.10.012. pmid: 37944488
[12]	Verma S, Das M, Sharma RD, et al. A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis. bioRxiv, 2023[2025-07-15]. doi:10.1101/2023.12.01.569475. Online ahead of print.
[13]	Subbarao S, Sanchez-Garrido J, Krishnan N, et al. Genetic and pharmacological inhibition of inflammasomes reduces the survival of Mycobacterium tuberculosis strains in macrophages. Sci Rep, 2020, 10(1): 3709. doi:10.1038/s41598-020-60560-y.
[14]	Kumar R, Singh P, Kolloli A, et al. Immunometabolism of Phagocytes During Mycobacterium tuberculosis Infection. Front Mol Biosci, 2019, 6: 105. doi:10.3389/fmolb.2019.00105.
[15]	王健宏, 徐兆坤, 李武. 炎性小体的激活机制及其在机体抗结核分枝杆菌中的作用研究进展. 畜牧兽医学报, 2020, 51(1):27-34. doi:10.11843/j.issn.0366-6964.2020.01.004.
[16]	Yi Z, Gao K, Li R, et al. Changed immune and miRNA response in RAW264.7 cells infected with cell wall deficient Mycobacterium tuberculosis. Int J Mol Med, 2018, 41(5): 2885-2892. doi:10.3892/ijmm.2018.3471.
[17]	张子扬. 结核分枝杆菌PknG对巨噬细胞极化的影响及其机制研究. 遵义: 遵义医科大学, 2022.

基因	正向引物	反向引物
白细胞介素-1β	5'-TCGCAGCAGCACATCAACAAGAG-3'	5'-TGCTCATGTCCTCATCCTGGAAGG-3'
肿瘤坏死因子α	5'-GGACTAGCCAGGAGGGAGAACAG-3'	5'-GCCAGTGAGTGAAAGGGACAGAAC-3'
白细胞介素-6	5'-ACTTCCATCCAGTTGCCTTCTTGG-3'	5'-TTAAGCCTCCGACTTGTGAAGTGG-3'
β-肌动蛋白	5'-GGCTGTATTCCCCTCCATCG-3'	5'-CCAGTTGGTAACAATGCCATGT-3'

菌株	培养0h(×10⁴CFU)	培养72h(×10⁴CFU)	存活率(%)
H37Rv-WT	5.20±0.31	2.13±0.11	41.04±2.11
H37RvΔRv1527c	5.15±0.28	0.68±0.05	13.13±1.05

炎症因子	时间点	H37Rv-WT	H37RvΔRv1527c	t值	P值
白细胞介素-1β	2h	1.05±0.08	1.52±0.11	3.245	0.023
	4h	1.82±0.12	3.15±0.22	9.874	0.001
	8h	2.15±0.16	4.88±0.30	12.587	<0.001
	12h	2.87±0.19	4.20±0.25	7.452	0.002
	24h	3.12±0.22	3.85±0.26	1.932	0.124
肿瘤坏死因子α	2h	1.08±0.09	1.25±0.10	1.874	0.143
	4h	1.12±0.10	1.98±0.15	4.562	0.010
	8h	1.95±0.15	2.35±0.18	2.114	0.104
	12h	2.05±0.16	3.12±0.21	5.328	0.006
	24h	2.87±0.20	3.25±0.23	1.745	0.168
白细胞介素-6	2h	1.02±0.07	0.95±0.06	1.245	0.285
	4h	1.15±0.09	1.02±0.08	1.587	0.198
	8h	1.87±0.13	1.70±0.12	1.332	0.264
	12h	2.45±0.18	2.20±0.16	1.547	0.212
	24h	3.15±0.22	1.42±0.10	11.732	<0.001

pks5基因(Rv1527c)缺失削弱结核分枝杆菌巨噬细胞内生存能力并调控炎症因子表达

Deletion of the pks5 gene (Rv1527c) impairs intracellular survival in macrophages and modulates inflammatory cytokine expression in Mycobacterium tuberculosis

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