Real-time probing of membrane transport in living microbial cells using single nanoparticle optics and living cell imaging
Real-time probing of membrane transport in living microbial cells using single nanoparticle optics and living cell imaging
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DOI：
10.1021/bi036231a
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发表时间：
2004-08-17
期刊：
影响因子：
2.9
通讯作者：
Viola, JJ
中科院分区：
文献类型：
--
作者：
Xu, XHN;Brownlow, WJ;Viola, JJ
膜运输在多种细胞和亚细胞途径中发挥着主导作用，包括多药耐药性 (MDR)、细胞信号传导和细胞间通讯。铜绿假单胞菌以其有趣的膜转运机制而闻名，例如膜渗透性和挤出机制的相互作用，导致特定细胞内物质的选择性积累和 MDR。尽管进行了广泛的研究，但活微生物细胞中的膜运输机制仍不完全清楚。在本研究中，我们利用银（Ag）纳米颗粒的固有颜色指数（表面等离子体共振光谱）作为纳米尺寸指数探针，直接测量纳米尺度上铜绿假单胞菌膜渗透性和孔径的实时变化。结果表明，尺寸最大为80 nm的银纳米颗粒在活微生物细胞中积累，证明这些银纳米颗粒可以穿过细胞的内膜和外膜。此外，随着氯霉素浓度的增加，在细胞中观察到更多数量的较大细胞内银纳米颗粒，这表明氯霉素增加了膜的通透性和孔隙率。此外，对突变体（nalB-1和DeltaABM）的研究表明，细胞内Ag纳米颗粒的积累速率取决于挤出泵（MexAB-OprM）的表达水平，这表明挤出泵在控制Ag纳米颗粒在活细胞中的积累中发挥着重要作用。此外，银纳米粒子测量的积累动力学与使用小荧光分子（EtBr）测量的相似，消除了银纳米粒子探针的空间和尺寸效应的可能性。敏感性测量还表明，低浓度的银纳米颗粒（1.3 pM）不会对细胞产生明显的毒性，进一步验证了单个银纳米颗粒（1.3 pM）可以用作生物相容性纳米探针，用于研究活微生物细胞的膜传输动力学。
Membrane transport plays a leading role in a wide spectrum of cellular and subcellular pathways, including multidrug resistance (MDR), cellular signaling, and cell-cell communication. Pseudomonas aeruginosa is renowned for its intriguing membrane transport mechanisms, such as the interplay of membrane permeability and extrusion machinery, leading to selective accumulation of specific intracellular substances and MDR. Despite extensive studies, the mechanisms of membrane transport in living microbial cells remain incompletely understood. In this study, we directly measure real-time change of membrane permeability and pore sizes of P. aeruginosa at the nanometer scale using the intrinsic color index (surface plasmon resonance spectra) of silver (Ag) nanoparticles as the nanometer size index probes. The results show that Ag nanoparticles with sizes ranging up to 80 nm are accumulated in living microbial cells, demonstrating that these Ag nanoparticles transport through the inner and outer membrane of the cells. In addition, a greater number of larger intracellular Ag nanoparticles are observed in the cells as chloramphenicol concentration increases, suggesting that chloramphenicol increases membrane permeability and porosity. Furthermore, studies of mutants (nalB-1 and DeltaABM) show that the accumulation rate of intracellular Ag nanoparticles depends on the expression level of the extrusion pump (MexAB-OprM), suggesting that the extrusion pump plays an important role in controlling the accumulation of Ag nanoparticles in living cells. Moreover, the accumulation kinetics measured by Ag nanoparticles are similar to those measured using a small fluorescent molecule (EtBr), eliminating the possibility of steric and size effects of Ag nanoparticle probes. Susceptibility measurements also suggest that a low concentration of Ag nanoparticles (1.3 pM) does not create significant toxicity for the cells, further validating that single Ag nanoparticles (1.3 pM) can be used as biocompatible nanoprobes for the study of membrane transport kinetics in living microbial cells.