Rv2048c基因敲除对结核分枝杆菌药物敏感性的影响研究

doi:10.19982/j.issn.1000-6621.20250429

中国防痨杂志 ›› 2025, Vol. 47 ›› Issue (12): 1621-1628.doi: 10.19982/j.issn.1000-6621.20250429

Rv2048c基因敲除对结核分枝杆菌药物敏感性的影响研究

李娜娜¹^,²(), 王鸿¹^,³, 田鹏¹, 文继秀¹, 杨丹¹, 吴霞¹, 兰远波¹()

¹遵义医科大学附属医院结核病区,遵义 563003
²贵州医科大学免疫教研室,贵阳 561113
³遵义市第一人民医院呼吸与危重症医学科,遵义 563099

收稿日期:2025-11-04 出版日期:2025-12-10 发布日期:2025-11-28
通信作者: 李娜娜,Email:252578718@qq.com;兰远波,Email:2987683018@qq.com
基金资助:
贵州省科技厅基础研究计划(黔科合基础-ZK[2022]一般648);贵州省卫生健康委科学技术基金(gzwkj2024-106);贵州省卫生健康委科学技术基金(gzwkj2021-101);国家自然科学基金(81860004)

Investigation into the impact of Rv2048c gene knockout on the drug susceptibility of Mycobacterium tuberculosis

Li Nana¹^,²(), Wang Hong¹^,³, Tian Peng¹, Wen Jixiu¹, Yang Dan¹, Wu Xia¹, Lan Yuanbo¹()

¹Department of Tuberculosis, Affiliated Hospital of Zunyi Medical University, Zunyi 563003, China
²Department of Immunology, Guizhou Medical University, Guiyang 561113, China
³Department of Respiratory and Critical Care Medicine, the First People's Hospital of Zunyi, Zunyi 563099, China

Received:2025-11-04 Online:2025-12-10 Published:2025-11-28
Contact: Li Nana, Email: 252578718@qq.com;Lan Yuanbo, Email: 2987683018@qq.com
Supported by:
Guizhou Provincial Research Foundation for Basic Research(Qiankehe jichu [2022] 648);Science and Technology Fund Project of Guizhou Provincial Health Commission(gzwkj2024-106);Science and Technology Fund Project of Guizhou Provincial Health Commission(gzwkj2021-101);National Natural Science Foundation of China(81860004)

摘要/Abstract

摘要：

目的: 探讨Rv2048c基因敲除对结核分枝杆菌(Mycobacterium tuberculosis, MTB)药物敏感性的影响。方法: 本研究以MTB标准菌株(H37Rv-WT)为模版,基于同源重组原理构建Rv2048c基因敲除株(H37RvΔRv2048c)。通过固体培养、抗酸染色、扫描电镜观察菌落形态、菌体大小、细胞壁完整性;绘制生长曲线评估生长速度;采用微孔板法药物敏感性试验测定最低抑菌浓度(minimum inhibitory concentration, MIC);利用溴化乙锭染色法检测细胞壁通透性。比较H37Rv-WT与H37RvΔRv2048c在生物学特性、抗结核药物敏感性及细胞壁通透性方面的差异,进而分析Rv2048c基因对MTB药物敏感性的作用及其潜在机制。结果: 成功构建了H37RvΔRv2048c;菌落形态由H37Rv-WT的粗糙型(R型)转变为H37RvΔRv2048c的光滑型(S型);两种菌体均呈棒状结构,细胞壁基本一致,H37RvΔRv2048c菌体长度变短;乙硫异烟胺(ethionamide, Eto)对H37RvΔRv2048c的MIC(1.25μg/ml)为Eto对H37Rv-WT的MIC(5.00μg/ml)的1/4;阿米卡星(amikacin, Am)对H37RvΔRv2048c的MIC(0.25μg/ml)为Am对H37Rv-WT的MIC(1.00μg/ml)的1/4;溴化乙锭染色后H37RvΔRv2048c的荧光强度(1.526RFU)较H37Rv-WT(0.924RFU)显著增高(t=16.960,P<0.001)。结论: Rv2048c基因的缺失可降低Eto和Am的MIC,该基因可能通过调控MTB细胞壁通透性影响MTB对Eto和Am的敏感性,为理解MTB耐药机制提供了新视角,并为增强Eto与Am疗效的研究提供了潜在靶点。

关键词: 分枝杆菌, 结核, 基因敲除, 微生物敏感性试验, 研究

Abstract:

Objective: To investigate the effect of Rv2048c gene knockout on the drug susceptibility of Mycobacterium tuberculosis (MTB). Methods: In this study, the MTB standard strain (H37Rv-WT) was used as a template to construct the Rv2048c gene knockout strain (H37RvΔRv2048c) via homologous recombination principle. Colony morphology, cell size, and cell wall integrity were evaluated through solid culture, acid-fast staining, and scanning electron microscopy. A growth curve was plotted to evaluate the growth rate. The minimum inhibitory concentration (MIC) was determined using the microplate method for drug susceptibility testing. Cell wall permeability was assessed using ethidium bromide staining. The biological characteristics, anti-tuberculosis drug susceptibility, and cell wall permeability of H37Rv-WT and H37RvΔRv2048c were systematically compared. Subsequently, the influence of the Rv2048c gene on MTB drug susceptibility and its potential mechanism were analyzed. Results: H37RvΔRv2048c was successfully constructed. Colony morphology transitioned from the rough type (R type) in H37RV-WT to the smooth type (S type) in H37RvΔRv2048c. Both strains exhibited rod-like structures with comparable cell wall compositions; however, the bacterial length of H37RvΔRv2048c was significantly shorter. The MIC of ethionamide (Eto) against H37RvΔRv2048c (1.25 μg/ml) was 1/4 of the MIC of Eto against H37Rv-WT (5.00 μg/ml); the MIC of amikacin (Am) against H37RvΔRv2048c (0.25 μg/ml) was 1/4 of the MIC of Am against H37Rv-WT (1.00 μg/ml). After ethidium bromide staining, the fluorescence intensity of H37RvΔRv2048c (1.526 RFU) was significantly higher than that of H37Rv-WT (0.924 RFU)(t=16.960, P<0.001). Conclusion: Deletion of the Rv2048c gene results in reduced MIC values for Eto and Am. This gene may modulate MTB susceptibility to Eto and Am by altering cell wall permeability. This study provides new insights into MTB drug resistance mechanisms and identifies potential targets for enhancing the effectiveness of Eto and Am.

Key words: Mycobacterium tuberculosis, Knockout, Microbial sensitivity tests, Research

中图分类号:

R521

李娜娜, 王鸿, 田鹏, 文继秀, 杨丹, 吴霞, 兰远波. Rv2048c基因敲除对结核分枝杆菌药物敏感性的影响研究[J]. 中国防痨杂志, 2025, 47(12): 1621-1628. doi: 10.19982/j.issn.1000-6621.20250429

Li Nana, Wang Hong, Tian Peng, Wen Jixiu, Yang Dan, Wu Xia, Lan Yuanbo. Investigation into the impact of Rv2048c gene knockout on the drug susceptibility of Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2025, 47(12): 1621-1628. doi: 10.19982/j.issn.1000-6621.20250429

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药物类别	MIC(μg/ml)
药物类别	H37Rv-WT	H37RvΔRv2048c
氧氟沙星	0.25	0.25
莫西沙星	<0.06	<0.06
利福平	<0.12	<0.12
阿米卡星	0.50	<0.12
链霉素	0.50	<0.25
利福布汀	<0.12	<0.12
对氨基水杨酸	0.50	0.50
乙硫异烟胺	20.00	0.60
环丝氨酸	4.00	4.00
异烟肼	0.06	<0.03
卡那霉素	1.20	<0.60
乙胺丁醇	1.00	<0.50

Rv2048c基因敲除对结核分枝杆菌药物敏感性的影响研究

Investigation into the impact of Rv2048c gene knockout on the drug susceptibility of Mycobacterium tuberculosis

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