Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Higashimurayama, Tokyo, Japan
Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Higashimurayama, Tokyo, Japan.
mSphere. 2021 Jan 6;6(1):e00518-20. doi: 10.1128/mSphere.00518-20.
The increasing occurrence of multidrug-resistant () is a serious threat to global public health. Among the many mechanisms of drug resistance, only resistance-nodulation-division (RND)-type multidrug efflux systems can simultaneously render bacteria tolerant to numerous toxic compounds, including antibiotics. The elevated expression of RND-type xenobiotic efflux transporter complexes, which consist of an inner membrane transporter, membrane fusion protein, and outer membrane channel, plays a major role in multidrug resistance. Among the 14 mycobacterial membrane protein large (MmpL) proteins identified as inner membrane transporters of , MmpL5 is known to participate in the acquisition of resistance to bedaquiline and clofazimine. MmpL5 exports these drugs by forming a complex with the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5). However, the role of MmpS5 in the efflux of antituberculous drugs by MmpL5 remains unclear. In this study, we focused on the dynamics of MmpL5 using green fluorescent protein (GFP). Single-molecule observations of MmpL5 showed substantial lateral displacements of MmpL5-GFP without the expression of MmpS5. Nondiffusing MmpL5-GFP foci typically showed three-step photobleaching, suggesting that MmpL5 formed a homotrimeric functional complex on the inner membrane in the presence of MmpS5. These results suggest that the expression of MmpS5 facilitates the assembly of monomeric MmpL5 into a homotrimer that is anchored to the inner membrane to transport various antimycobacterial drugs. It has been reported that mycobacterial membrane protein large 5 (MmpL5), a resistance-nodulation-division (RND)-type inner membrane transporter in (), is involved in the transport of antimycobacterial drugs. However, the functional roles of the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5), organized as an operon with MmpL5, are unclear. Via the single-molecule imaging of MmpL5, we uncovered the maintenance of the functional trimeric complex structure of MmpL5 in the presence of MmpS5. These findings demonstrate that the assembly mechanisms of mycobacterial RND efflux systems are the dynamically regulated process through interactions among the components. This represents the first report of the single-molecule observation of efflux transporters, which may enhance our understanding of innate antibiotic resistance.
越来越多的多药耐药 () 的出现对全球公共健康构成了严重威胁。在许多耐药机制中，只有耐药性结节分裂 (RND) 型多药外排系统能够使细菌同时耐受多种有毒化合物，包括抗生素。RND 型外排转运蛋白复合物的高表达，该复合物由内膜转运蛋白、膜融合蛋白和外膜通道组成，在多药耐药中起主要作用。在 14 种鉴定为 的分枝杆菌膜蛋白大 (MmpL) 蛋白中，MmpL5 已知参与获得对贝达喹啉和氯法齐明的耐药性。MmpL5 通过与膜融合蛋白分枝杆菌膜蛋白小 5 (MmpS5) 形成复合物来排出这些药物。然而，MmpS5 在 MmpL5 排出抗结核药物中的作用尚不清楚。在这项研究中，我们专注于使用绿色荧光蛋白 (GFP) 观察 MmpL5 的 动力学。MmpL5-GFP 的单分子观察显示，在没有表达 MmpS5 的情况下，MmpL5 发生了大量的横向位移。非扩散的 MmpL5-GFP 焦点通常表现出三步光漂白，表明在存在 MmpS5 的情况下，MmpL5 在内膜上形成三聚体功能复合物。这些结果表明，MmpS5 的表达促进了单体 MmpL5 组装成三聚体，该三聚体锚定在内膜上以转运各种抗分枝杆菌药物。据报道，分枝杆菌膜蛋白大 5 (MmpL5) 是分枝杆菌 () 中的一种耐药性结节分裂 (RND) 型内膜转运蛋白，参与抗分枝杆菌药物的转运。然而，与 MmpL5 作为操纵子组织的膜融合蛋白分枝杆菌膜蛋白小 5 (MmpS5) 的功能作用尚不清楚。通过 MmpL5 的单分子成像，我们揭示了在存在 MmpS5 的情况下，MmpL5 的功能性三聚体复合物结构的维持。这些发现表明，分枝杆菌 RND 外排系统的组装机制是通过各组件之间的相互作用进行动态调节的过程。这是首次报道分枝杆菌外排转运蛋白的单分子观察，可能有助于我们更好地理解先天抗生素耐药性。
