Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Seongnam-si, South Korea.
mSystems. 2024 Jan 23;9(1):e0085123. doi: 10.1128/msystems.00851-23. Epub 2023 Dec 19.
Artemisinin (ARS) displayed bactericidal activity against . To assess the mechanistic details of its antibacterial action, we have isolated mutants with enhanced ARS resistance and identified a gene (VCA0767) whose loss-of-function resulted in the ARS resistance phenotypes. This gene () encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding a esistance-odulation-cell ivision (RND) efflux pump operon () and the outer membrane component () were highly upregulated in the mutant, suggesting that AtrR might act as a negative regulator of this operon and . Gene deletion of , , or abrogated the ARS resistance of the mutant, and more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the mutant. The cytoplasmic accumulation of ARS was compromised in the mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.IMPORTANCEDrug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a esistance-odulation-cell ivision (RND) efflux pump of , is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during pathogenesis.
青蒿素 (ARS) 对 表现出杀菌活性。为了评估其抗菌作用的机制细节，我们分离出了对 ARS 具有增强抗性的 突变体，并鉴定出一个基因 (VCA0767)，其功能丧失导致了 ARS 抗性表型。该基因 () 编码一个 TetR 家族转录调节因子，其缺失突变体显示 ARS 诱导的 ROS 形成和 DNA 损伤减少。转录组分析显示，编码一个 耐药性调节细胞分裂 (RND) 外排泵操纵子 () 和外膜组件 () 的基因在 突变体中高度上调，表明 AtrR 可能作为该操纵子的负调节剂。该基因的缺失 () 、 或 消除了 突变体的 ARS 抗性，更重要的是，VexAB-TolC 的异位表达足以赋予 ARS 抗性，表明 VexAB-TolC 外排系统的表达增加对于 突变体的 ARS 抗性是必要和充分的。ARS 在 突变体中的细胞质积累受到损害，表明 VexAB-TolC 可能是将 ARS 排出到细菌细胞内以降低其毒性的主要外排系统。在 VexR 依赖的方式下， 突变体对红霉素也表现出抗性。该结果表明，AtrR 可能作为一种全局调节剂发挥作用，通过增强 RND 外排系统来防止细胞内积累有毒化学物质。重要的是，尽管增加表达外排泵被认为是细菌病原体中多种抗生素耐药性的主要决定因素，但它们的底物特异性和调节机制仍知之甚少。在这里，我们证明了 VexAB-TolC，一种 的 耐药性调节细胞分裂 (RND) 外排泵，负责对抗疟药物青蒿素 (ARS) 的耐药性，ARS 具有杀菌活性。此外，我们新鉴定出 AtrR，一种 TetR 家族阻遏物，作为 VexRAB 和通用外膜通道 TolC 的全局调节剂，其中 VexR 作为 vexAB 操纵子的途径特异性调节剂发挥作用。我们的发现将有助于提高我们对 VexAB-TolC 系统广泛的底物特异性的认识，并强调了在 发病机制过程中多种 RND 外排系统的复杂调控网络。
