Zhang Han, Fang Cuiting, Yusuf Buhari, Zhu Xiaoqing, Wang Shuai, Hameed H M Adnan, Gao Yamin, Zhang Tianyu
School of Basic Medical Sciences, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230000, China.
State Key Laboratory of Respiratory Disease, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510000, China.
Int J Mol Sci. 2025 Apr 17;26(8):3779. doi: 10.3390/ijms26083779.
The emergence of multidrug-resistant and extensively drug-resistant strains poses serious challenges to global tuberculosis control, highlighting the urgent need to elucidate the mechanisms underlying multidrug resistance. In this study, we screened for spontaneous bortezomib (BTZ)-resistant (Msm) mutants and identified a strain, Msm-R1-2, exhibiting 16- and 64-fold increases in minimum inhibitory concentrations (MICs) to BTZ and linezolid (LZD), respectively, compared to the parental strain. Whole-genome sequencing revealed resistance-associated mutations in two functionally distinct genes: , encoding a transcriptional regulator involved in efflux pump expression, and , encoding a porin protein. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that single mutations in either or caused low-level resistance (4-fold MIC increase) to BTZ and LZD, while dual mutations conferred resistance levels comparable to Msm-R1-2, with 16- and 64-fold increases in MICs for BTZ and LZD, respectively. An ethidium bromide accumulation assay demonstrated that mutations in reduce cell wall permeability, contributing to multidrug resistance. Furthermore, quantitative real-time PCR showed that mutations in upregulate the - efflux system. Together, these dual mechanisms function synergistically: restricted drug entry combined with enhanced drug efflux confers robust multidrug resistance. These findings provide novel insights into the evolutionary mechanisms of resistance in mycobacteria.
多重耐药和广泛耐药菌株的出现给全球结核病控制带来了严峻挑战，凸显了阐明多重耐药潜在机制的迫切需求。在本研究中，我们筛选了自发的硼替佐米（BTZ）耐药（耻垢分枝杆菌）突变体，并鉴定出一株耻垢分枝杆菌R1-2菌株，与亲本菌株相比，其对BTZ和利奈唑胺（LZD）的最低抑菌浓度（MIC）分别增加了16倍和64倍。全基因组测序揭示了两个功能不同基因中的耐药相关突变：一个编码参与外排泵表达的转录调节因子，另一个编码孔蛋白。CRISPR-Cpf1辅助的基因敲除和编辑实验证实，任一基因的单突变导致对BTZ和LZD的低水平耐药（MIC增加4倍），而双突变赋予的耐药水平与耻垢分枝杆菌R1-2相当，BTZ和LZD的MIC分别增加16倍和64倍。溴化乙锭积累试验表明，该基因的突变降低了细胞壁通透性，导致多重耐药。此外，定量实时PCR显示，该基因的突变上调了外排系统。总之，这两种双重机制协同发挥作用：药物进入受限与药物外排增强共同导致强大的多重耐药性。这些发现为分枝杆菌耐药的进化机制提供了新的见解。
