Single live cell imaging for real-time monitoring of resistance mechanism in Pseudomonas aeruginosa
Single live cell imaging for real-time monitoring of resistance mechanism in Pseudomonas aeruginosa
复制标题
DOI：
10.1117/1.1506707
复制
发表时间：
2002-10-01
影响因子：
3.5
通讯作者：
Xu, XHN
中科院分区：
文献类型：
--
作者：
Kyriacou, SV;Nowak, ME;Xu, XHN
我们已经开发并应用单活细胞成像实时监测铜绿假单胞菌的耐药动力学。在特定底物（EtBr）存在下的活细胞的实时图像提供了具有空间和时间信息的抗性机制的第一个直接见解，并且显示底物似乎在细胞质空间中积累，而不是周质空间。使用铜绿假单胞菌的三种突变体PAO 4290（MexAB-OprM的野生型表达水平）、TNP 030#1（nalB-1，MexAB-OprM过表达突变体）和TNP 076（DeltaABM，MexAB-OprM缺陷突变体）来研究这三种膜蛋白（MexAB-OprM）在抗性机制中的作用。选择溴化乙锭（EtBr）作为荧光探针，用于体细胞溶液的光谱测量和体细胞的单细胞成像。体积测量表明，在三个突变体中，nalB-1积累最少的EtBr和表现出最高的抗性EtBr，而DeltaABM积累最多的EtBr和表现出最低的抗性EtBr。这一结果表明MexAB-OprM蛋白通过将EtBr挤出细胞而在抗性机制中发挥作用。与整体测量不同，在单细胞分辨率下对整体细胞的成像和分析表明单个细胞具有其独特的抗性动力学，并提供了具有空间和时间分辨率的EtBr流入和流出调节的直接观察。与荧光染色分析不同，活细胞成像提供了三种突变体的膜通透性和外排泵机制的实时动力学信息。这项研究首次以单细胞分辨率直接成像活细菌细胞的耐药机制，为推进细菌耐药机制的理解开辟了新的可能性。(C)2002年，美国光电仪器工程师学会。
We have developed and applied single live cell imaging for real-time monitoring of resistance kinetics of Pseudomonas aeruginosa. Real-time images of live cells in the presence of a particular substrate (EtBr) provided the first direct insights of resistance mechanism with both spatial and temporal information and showed that the substrate appeared to be accumulated in cytoplasmic space, but not periplasmic space. Three mutants of P. aeruginosa, PAO4290 (a wildtype expression level of MexAB-OprM), TNP030#1 (nalB-1, MexAB-OprM over expression mutant), and TNP076 (DeltaABM, MexAB-OprM deficient mutant), were used to investigate the roles of these three membrane proteins (MexAB-OprM) in the resistance mechanism. Ethidium bromide (EtBr) was chosen as a fluorescence probe for spectroscopic measurement of bulk cell solution and single cell imaging of bulk cells. Bulk measurement indicated, among three mutants, that nalB-1 accumulated the least EtBr and showed the highest resistance to EtBr, whereas DeltaABM accumulated the most EtBr and showed the lowest resistance to EtBr. This result demonstrated the MexAB-OprM proteins played the roles in resistance mechanism by extruding EtBr out of cells. Unlike the bulk measurement, imaging and analysis of bulk cells at single cell resolution demonstrated individual cell had its distinguished resistance kinetics and offered the direct observation of the regulation of influx and efflux of EtBr with both spatial and temporal resolution. Unlike fluorescent staining assays, live cell imaging provided the real-time kinetic information of transformation of membrane permeability and efflux pump machinery of three mutants. This research constitutes the first direct imaging of resistance mechanism of live bacterial cells at single cell resolution and opens up the new possibility of advancing the understanding of bacteria resistance mechanism. (C) 2002 Society of Photo-Optical Instrumentation Engineers.