Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529, USA.
Biochemistry. 2010 Jul 20;49(28):5942-53. doi: 10.1021/bi100268k.
Multidrug membrane transporters (efflux pumps) in both prokaryotes and eukaryotes are responsible for impossible treatments of a wide variety of diseases, including infections and cancer, underscoring the importance of better understanding of their structures and functions for the design of effective therapies. In this study, we designed and synthesized two silver nanoparticles (Ag NPs) with average diameters of 13.1 +/- 2.5 nm (8.1-38.6 nm) and 91.0 +/- 9.3 nm (56-120 nm) and used the size-dependent plasmonic spectra of single NPs to probe the size-dependent transport kinetics of MexAB-OprM (multidrug transporter) in Pseudomonas aeruginosa in real time at nanometer resolution. We found that the level of accumulation of intracellular NPs in wild-type (WT) cells was higher than in nalB1 (overexpression of MexAB-OprM) but lower than in DeltaABM (deletion of MexAB-OprM). In the presence of proton ionophores (CCCP, inhibitor of proton motive force), we found that intracellular NPs in nalB1 were nearly doubled. These results suggest that MexAB-OprM is responsible for the extrusion of NPs out of cells and NPs (orders of magnitude larger than conventional antibiotics) are the substrates of the transporter, which indicates that the substrates may trigger the assembly of the efflux pump optimized for the extrusion of the encountered substrates. We found that the smaller NPs stayed inside the cells longer than larger NPs, suggesting the size-dependent efflux kinetics of the cells. This study shows that multisized NPs can be used to mimic various sizes of antibiotics for probing the size-dependent efflux kinetics of multidrug membrane transporters in single living cells.
多药膜转运蛋白（外排泵）在原核生物和真核生物中都负责治疗多种疾病，包括感染和癌症，这凸显了更好地了解其结构和功能对于设计有效治疗方法的重要性。在这项研究中，我们设计并合成了两种平均直径分别为 13.1±2.5nm（56-38.6nm）和 91.0±9.3nm（56-120nm）的银纳米粒子（Ag NPs），并利用单 NPs 的尺寸依赖等离子体光谱实时探测铜绿假单胞菌中 MexAB-OprM（多药转运蛋白）的尺寸依赖转运动力学。我们发现，野生型（WT）细胞内 NPs 的积累水平高于 nalB1（MexAB-OprM 的过表达），但低于 DeltaABM（MexAB-OprM 的缺失）。在质子离子载体（CCCP，质子动力势抑制剂）存在的情况下，我们发现 nalB1 细胞内的 NPs 几乎增加了一倍。这些结果表明，MexAB-OprM 负责将 NPs 从细胞中排出，而 NPs（比传统抗生素大几个数量级）是转运体的底物，这表明底物可能触发了优化用于排出遇到的底物的外排泵的组装。我们发现，较小的 NPs 在细胞内停留的时间比较大的 NPs 长，这表明细胞的尺寸依赖外排动力学。本研究表明，多尺寸 NPs 可用于模拟各种抗生素的大小，以探测单活细胞中多药膜转运蛋白的尺寸依赖外排动力学。
