脓肿分枝杆菌（Mycobacterium abscessus）是一种机会性致病菌，可引起人类和动物多种感染，近年来引起的肺部、皮肤软组织及免疫缺陷患者播散性感染显著增多。该菌兼具天然耐药性和强致病性特征，导致临床治疗困难，亟待阐明其耐药机制与致病因子。研究表明，外排泵系统可能通过外排抗生素介导耐药性，还可通过调控糖肽脂合成、生物膜形成及免疫逃逸等途径间接参与致病过程。尽管脓肿分枝杆菌拥有两百多个外排泵编码基因，但这些基因在脓肿分枝杆菌的耐药和致病中的作用尚不明确。

为探究脓肿分枝杆菌外排泵基因的耐药机制及其致病作用，基于前期构建的68个外排泵突变株，我们通过转入条形码（Barcode）序列标记质粒成功建立突变文库。将该文库感染大蜡螟幼虫模型，筛选得到多个与致病力相关的外排泵基因。选择MAB_3871c和MAB_3142c两个关键基因进行功能验证，发现其突变可显著削弱细菌在幼虫体内的毒力，其中MAB_3142c缺失株的致病性降低更为显著，提示该基因对脓肿分枝杆菌的致病力更关键。通过最低抑菌浓度检测、药敏片扩散和梯度稀释滴板实验，我们发现MAB_3142c的突变增强了脓肿分枝杆菌对阿米卡星、贝达喹啉、克拉霉素和利奈唑胺的敏感性。进一步研究发现MAB_3142c的突变不影响脓肿分枝杆菌中溴化乙锭的积累，提示MAB_3142c可能并非通过影响脓肿分枝杆菌的细胞壁通透性介导其天然耐药。

综上，本研究利用大蜡螟幼虫模型系统研究了外排泵对脓肿杆菌致病性的影响，发现了脓肿分枝杆菌外排泵MAB_3142c在耐药与致病中的双重作用。该研究初步揭示了外排泵在脓肿分枝杆菌耐药致病中的作用，为深入解析其功能及作用机制奠定了研究基础，也为研究新型抗脓肿分枝杆菌感染的药物提供了靶标。

Mycobacterium abscessus (M.abs) is an opportunistic pathogen that can cause a variety of infections in humans and animals. In recent years, the incidence of infections caused by this pathogen, including pulmonary infections, skin and soft tissue infections, and disseminated infections in immunocompromised patients, has risen significantly. Given its intrinsic drug resistance and significant virulence, clinical treatment faces substantial challenges, making in-depth research into its drug resistance mechanisms and virulence factors particularly urgent. Research has demonstrated that efflux pump systems may mediate drug resistance via antibiotic extrusion and may contribute to pathogenicity by regulating glycopeptidolipid synthesis, biofilm formation, and immune evasion. Despite harboring more than 200 efflux pump genes, their specific roles in M. abscessus drug resistance and virulence remain unclear.

To thoroughly investigate the drug resistance mechanisms mediated by efflux pump genes in M. abscessus and their role in pathogenicity, we developed a comprehensive mutant library comprising 68 efflux pump deletion strains, each tagged with unique barcode sequences. Screening in the Galleria mellonella larva infection model identified MAB_3871cand MAB_3142c as critical virulence determinants, with deletion mutants exhibiting significantly impaired larval colonization capacity. Subsequent validation experiments revealed that ΔMAB_3142c infection resulted in higher larval survival compared to wild-type, establishing its essential role in pathogenicity. Additionally, through experiments such as minimum inhibitory concentration testing, disk diffusion, and gradient dilution plating, we confirmed that MAB_3142cenhances the resistance of M. abscessus to amikacin, bedaquiline, clarithromycin, and linezolid. Intriguingly, ethidium bromide accumulation assays showed no significant differences between the ΔMAB_3142c and wild-type strains, suggesting an alternative, membrane permeability-independent resistance mechanism.

In summary, this study elucidates the dual role of efflux pump gene MAB_3142c in both drug resistance and pathogenicity of M. abscessus, demonstrating its potential value as a therapeutic target.  Furthermore, the constructed mutant library not only provides a robust platform for high-throughput screening of novel antimicrobial compounds, but also establishes a fundamental framework for investigating the role of efflux pumps in host-pathogen interactions.