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

doi:10.19983/j.issn.2096-8493.20250101

Abstract

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

CLC Number:

R378.91

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

Figures/Tables 5

References 17

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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