The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis 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 Mycobacterium smegmatis (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: MSMEG_1380, encoding a transcriptional regulator involved in efflux pump expression, and MSMEG_0965, encoding a porin protein. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that single mutations in either MSMEG_1380 or MSMEG_0965 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 MSMEG_0965 reduce cell wall permeability, contributing to multidrug resistance. Furthermore, quantitative real-time PCR showed that mutations in MSMEG_1380 upregulate the mmpS5-mmpL5 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.

The emergence of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains poses serious challenges to global tuberculosis control, highlighting the urgent need to unravel the mechanisms underlying multidrug resistance. In this study, we screened for spontaneous bortezomib (BTZ)-resistant mutants of Mycobacterium smegmatis and identified a strain, Msm-R1-2, which exhibited high-level resistance to both BTZ and linezolid. Whole-genome sequencing revealed mutations in MSMEG_1380 and MSMEG_0965 genes, encoding a transcriptional regulator (involved in regulating efflux pump expression) and a porin, respectively are potential contributors to drug resistance. CRISPR-Cpf1-assisted gene knockout and editing experiments confirmed that dual mutations in MSMEG_1380 and MSMEG_0965 synergistically enhanced resistance to BTZ and LZD, conferring cross-resistance to other antibiotics, including moxifloxacin and clofazimine. Ethidium bromide accumulation assay demonstrated that mutations in MSMEG_0965 reduce cell wall permeability, contributing to multidrug resistance. Furthermore, previous studies have shown that mutations in MSMEG_1380 upregulate the mmpS5-mmpL5 efflux system, thereby promoting drug efflux and reducing intracellular drug concentrations, while mutations in MSMEG_0965 impair porin function, limiting antibiotic uptake and significantly contributing to the multidrug-resistant phenotype. Collectively, these findings provide valuable insights into the molecular mechanisms of mycobacterial drug resistance, underscoring the pivotal roles of efflux and uptake pathways in the development of multidrug resistance.