Dual Mutations in MSMEG_0965 and MSMEG_1380 Confer High-Level Resistance to Bortezomib and Linezolid by Both Reducing Drug Intake and Increasing Efflux in <em>Mycobacterium smegmatis</em>

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.