Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, USA.
Analyst. 2018 Mar 26;143(7):1599-1608. doi: 10.1039/c7an01787a.
ATP-binding cassette (ABC) membrane transporters serve as self-defense transport apparatus in many living organisms and they can selectively extrude a wide variety of substrates, leading to multidrug resistance (MDR). The detailed molecular mechanisms remain elusive. Single nanoparticle plasmonic spectroscopy highly depends upon their sizes, shapes, chemical and surface properties. In our previous studies, we have used the size-dependent plasmonic spectra of single silver nanoparticles (Ag NPs) to study the real-time efflux kinetics of the ABC (BmrA) transporter and MexAB-OprM transporter in single live cells (Gram-positive and Gram-negative bacterium), respectively. In this study, we prepared and used purified, biocompatible and stable (non-aggregated) gold nanoparticles (Au NPs) (12.4 ± 0.9 nm) to study the efflux kinetics of single BmrA membrane transporters of single live Bacillus subtillis cells, aiming to probe chemical dependent efflux functions of BmrA transporters and their potential chemical sensing capability. Similar to those observed using Ag NPs, accumulation of the intracellular Au NPs in single live cells (WT and ΔBmrA) highly depends upon the cellular expression of BmrA and the NP concentration (0.7 and 1.4 nM). The lower accumulation of intracellular Au NPs in WT (normal expression of BmrA) than ΔBmrA (deletion of bmrA) indicates that BmrA extrudes the Au NPs out of the WT cells. The accumulation of Au NPs in the cells increases with NP concentration, suggesting that the Au NPs most likely passively diffuse into the cells, similar to antibiotics. The result demonstrates that such small Au NPs can serve as imaging probes to study the efflux function of the BmrA membrane transporter in single live cells. Furthermore, the dependence of the accumulation rate of intracellular Au NPs in single live cells upon the expression of BmrA and the concentration of the NPs is about twice higher than that of the same sized Ag NPs. This interesting finding suggests the chemical-dependent efflux kinetics of BmrA and that BmrA could distinguish nearly identical sized Au NPs from Ag NPs and might possess chemical sensing machinery.
三磷酸腺苷结合盒（ABC）膜转运蛋白作为许多生物的自我防御转运装置，可以选择性地外排多种底物，导致多药耐药（MDR）。其详细的分子机制仍不清楚。单纳米颗粒等离子体光谱高度依赖于它们的大小、形状、化学和表面性质。在我们之前的研究中，我们使用单银纳米颗粒（Ag NPs）的尺寸依赖等离子体光谱来分别研究 ABC（BmrA）转运蛋白和 MexAB-OprM 转运蛋白在单个活细胞（革兰氏阳性和革兰氏阴性菌）中的实时外排动力学。在这项研究中，我们制备并使用了纯化的、生物相容的和稳定的（非聚集的）金纳米颗粒（Au NPs）（12.4 ± 0.9nm）来研究单个活枯草芽孢杆菌细胞中单 BmrA 膜转运蛋白的外排动力学，旨在探测 BmrA 转运蛋白的化学依赖外排功能及其潜在的化学传感能力。与使用 Ag NPs 观察到的情况类似，细胞内 Au NPs 的积累高度依赖于 BmrA 转运蛋白的细胞表达和 NP 浓度（0.7 和 1.4nM）。WT（BmrA 正常表达）细胞内 Au NPs 的积累低于 ΔBmrA（bmrA 缺失）细胞，这表明 BmrA 将 Au NPs 从 WT 细胞中排出。细胞内 Au NPs 的积累随 NP 浓度的增加而增加，表明 Au NPs 很可能被动扩散进入细胞，类似于抗生素。结果表明，如此小的 Au NPs 可以作为成像探针来研究单个活细胞中 BmrA 膜转运蛋白的外排功能。此外，单个活细胞内 Au NPs 积累率对 BmrA 表达和 NPs 浓度的依赖性比相同尺寸的 Ag NPs 高约两倍。这一有趣的发现表明 BmrA 具有化学依赖的外排动力学，并且 BmrA 可以区分几乎相同尺寸的 Au NPs 和 Ag NPs，并可能具有化学传感机制。
