De Siena Barbara, Campolattano Nicoletta, D'Abrosca Gianluca, Russo Luigi, Cantillon Daire, Marasco Rosangela, Muscariello Lidia, Waddell Simon J, Sacco Margherita
Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Università degli Studi della Campania Luigi Vanvitelli, Caserta, Italy.
Department of Global Health and Infection, Brighton and Sussex Medical School, University of Sussex, Brighton, United Kingdom.
Front Microbiol. 2020 Dec 3;11:575828. doi: 10.3389/fmicb.2020.575828. eCollection 2020.
Multi-drug resistant tuberculosis (MDR-TB) represents a major health problem worldwide. Drug efflux and the activity of efflux transporters likely play important roles in the development of drug-tolerant and drug-resistant mycobacterial phenotypes. This study is focused on the action of a mycobacterial efflux pump as a mechanism of drug resistance. Previous studies demonstrated up-regulation of the TetR-like transcriptional regulator in and its ortholog in () in acid-nitrosative stress conditions. MSMEG-3765 regulates the expression of the operon, and of the orthologous region in (). MSMEG-3762 and Rv1687c are annotated as ATP-binding proteins, while MSMEG-3763 and Rv1686c are annotated as trans-membrane polypeptides, defining an ABC efflux pump in both and . The two putative efflux systems share a high percentage of identity. To examine the role of the putative efflux system MSMEG-3762/63, we constructed and characterized a deletion mutant in (∆). By comparative analysis of wild type, knockout, and complemented strains, together with structural modeling and molecular docking bioinformatics analyses of the MSMEG-3763 trans-membrane protein, we define the protein complex MSMEG-3762/63 as an efflux pump. Moreover, we demonstrate involvement of this pump in biofilm development and in the extrusion of rifampicin and ciprofloxacin (CIP), antimicrobial drugs used in first- and second-line anti-TB therapies.
耐多药结核病（MDR-TB）是全球主要的健康问题。药物外排及外排转运蛋白的活性可能在耐药物和耐药分枝杆菌表型的形成中发挥重要作用。本研究聚焦于分枝杆菌外排泵作为耐药机制的作用。先前的研究表明，在酸亚硝化应激条件下，结核分枝杆菌中TetR样转录调节因子及其在耻垢分枝杆菌中的直系同源物（MSMEG）会上调。MSMEG-3765调节结核分枝杆菌中mmpL3操纵子以及耻垢分枝杆菌中直系同源区域的表达。MSMEG-3762和Rv1687c被注释为ATP结合蛋白，而MSMEG-3763和Rv1686c被注释为跨膜多肽，在结核分枝杆菌和耻垢分枝杆菌中均定义了一个ABC外排泵。这两个假定的外排系统具有很高的同一性百分比。为了研究假定的外排系统MSMEG-3762/63的作用，我们构建并鉴定了耻垢分枝杆菌中的ΔmmpL3/mmpS3缺失突变体。通过对野生型、敲除型和互补型菌株的比较分析，以及对MSMEG-3763跨膜蛋白的结构建模和分子对接生物信息学分析，我们将蛋白复合物MSMEG-3762/63定义为一个外排泵。此外，我们证明了该泵参与生物膜形成以及利福平（RIF）和环丙沙星（CIP）的外排，这两种抗菌药物分别用于一线和二线抗结核治疗。
