Zhang Huiyun, Wang Shiyong, Zhang Yu, Hua Wenya, Lin Siran, Chen Xinchang, Xu Tao, Chen Jiazhen, Zhang Wenhong
Department of Infectious Diseases, Shanghai Key Laboratory of Infectious Diseases and Biosafety Emergency Response, National Medical Center for Infectious Diseases, Huashan Hospital, Shanghai Medical College, Fudan University, China.
Shanghai Sci-Tech Inno Center for Infection & Immunity, Shanghai, China.
PLoS Pathog. 2025 May 30;21(5):e1013190. doi: 10.1371/journal.ppat.1013190. eCollection 2025 May.
Mycobacterium abscessus (MAB) is a clinically significant multidrug-resistant (MDR) pathogen, particularly implicated in pulmonary infections among cystic fibrosis (CF) patients. Tedizolid (TZD), an oxazolidinone-class antibacterial drug, has been recommended as an alternative treatment for MAB-infected patients who are intolerant to or whose isolate is resistant to first-line drugs including linezolid (LZD). To investigate the TZD resistance mechanisms in MAB, we isolated 23 TZD-resistant MAB mutants and performed whole-genome sequencing (WGS) to identify resistance-associated genes. Frequent mutations were identified in MAB_2885, encoding a putative TetR transcriptional regulator, and MAB_2303, encoding a putative mycobacterial membrane protein large (MmpL). Drug susceptibility testing confirmed that MAB_2885 mutations contribute to both TZD and LZD resistance in MAB. RNA-seq analysis revealed that restoring wild-type MAB_2885 in mutants downregulated the MAB_2302-MAB_2303. Electrophoretic mobility shift assay (EMSA) showed the MAB_2885 protein binds to its target sequence upstream of MAB_2302-MAB_2303, further confirming their regulatory relationship. The W91R mutation in the MAB_2885 protein was found to impair its DNA-binding activity compared to the wild-type. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed that MAB_2302-MAB_2303 functions as a TZD efflux pump. Additionally, overexpression of MAB_2885 in M. abscessus subsp. bolletii and M. abscessus subsp. massiliense also increased their TZD susceptibility and downregulated their respective MmpS-MmpL orthologs. Overall, our study demonstrates that mutations in MAB_ 2885 contribute to TZD and LZD resistance by disrupting the negative regulation of the downstream MAB_2302-MAB_2303, which functions as a direct efflux pump for TZD. These findings provide new insights into oxazolidinone resistance mechanisms in MAB and identify potential biomarkers for detecting drug resistance.
脓肿分枝杆菌（MAB）是一种具有临床意义的多重耐药（MDR）病原体，尤其与囊性纤维化（CF）患者的肺部感染有关。特地唑胺（TZD）是一种恶唑烷酮类抗菌药物，已被推荐作为对包括利奈唑胺（LZD）在内的一线药物不耐受或其分离株耐药的MAB感染患者的替代治疗药物。为了研究MAB中TZD的耐药机制，我们分离出23株对TZD耐药的MAB突变体，并进行全基因组测序（WGS）以鉴定耐药相关基因。在编码假定的TetR转录调节因子的MAB_2885和编码假定的分枝杆菌膜蛋白大蛋白（MmpL）的MAB_2303中发现了频繁的突变。药敏试验证实，MAB_2885突变导致MAB对TZD和LZD均耐药。RNA测序分析表明，在突变体中恢复野生型MAB_2885会下调MAB_2302 - MAB_2303。电泳迁移率变动分析（EMSA）表明，MAB_2885蛋白与其在MAB_2302 - MAB_2303上游的靶序列结合，进一步证实了它们的调控关系。与野生型相比，发现MAB_2885蛋白中的W91R突变损害了其DNA结合活性。液相色谱 - 串联质谱（LC - MS/MS）分析证实，MAB_2302 - MAB_2303作为TZD外排泵发挥作用。此外，在脓肿分枝杆菌博列亚种和脓肿分枝杆菌马赛亚种中过表达MAB_2885也增加了它们对TZD的敏感性，并下调了它们各自的MmpS - MmpL直系同源物。总体而言，我们的研究表明，MAB_2885中的突变通过破坏下游作为TZD直接外排泵的MAB_2302 - MAB_2303的负调控，导致对TZD和LZD耐药。这些发现为MAB中恶唑烷酮耐药机制提供了新的见解，并确定了检测耐药性的潜在生物标志物。
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