Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-OprM drug extrusion machinery in Pseudomonas aeruginosa
Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-OprM drug extrusion machinery in Pseudomonas aeruginosa
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DOI：
10.1074/jbc.272.35.21964
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发表时间：
1997-08-29
影响因子：
4.8
通讯作者：
Nakae, T
中科院分区：
文献类型：
--
作者：
Ocaktan, A;Yoneyama, H;Nakae, T
铜绿假单胞菌的 MexA-MexB-OprM 外排泵由两种内膜蛋白 MexA 和 MexB 以及一种外膜蛋白 OprM 组成。我们通过评估删除这些亚基成分对几种荧光探针积累的影响，研究了该药物挤出系统的成分的作用，带正电的 2-(4-二甲基氨基苯乙烯基)-1-乙基吡啶鎓的荧光强度与野生型菌株相比，在缺乏OprM的突变体中，荧光强度和不带电的N-苯基-1-萘胺分别高出7倍和4倍，在缺乏MexA或MexB的突变体中，荧光强度分别高出4倍和1.7倍。这种荧光强度顺序与先前报道的抗生素（如四环素、氯霉素和氟喹诺酮类）的最低抑制浓度完全一致，所有菌株中溴化乙锭的积累Mex突变体的进展速度比野生型细胞快约5倍，该结果与β-内酰胺抗生素的最低抑制浓度一致。这些结果表明荧光探针可以成功地用于实时监测革兰氏阴性细菌中药物挤出机械的功能，1-(4-三甲基铵苯基)-6-苯基-1,3,5-己三烯从缺乏挤出泵的预装细胞中垂直于细胞质膜内叶定向的下坡挤出动力学在荧光强度缓慢增加之前，而野生型细胞立即释放染料，这一观察结果是这些结果反映了突变体中探针在细胞质膜中的较高积累，并强化了由 MexA-MexB-OprM 介导的疏水底物的挤出主要从细胞质膜内部发生的假设。
The MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa consists of two inner membrane proteins, MexA and MexB, and one outer membrane protein, OprM, We investigated the role of the components of this drug extrusion system by evaluating the repercussions of deleting these subunit components on the accumulation of several fluorescent probes, Fluorescence intensities of positively charged 2-(4-dimethylaminostyryl)-1-ethylpyridinium and uncharged N-phenyl-1-naphtylamine were 7 and 4 times higher, respectively, in the mutant lacking OprM and 4 and 1.7 times higher, respectively, in the mutants lacking MexA or MexB than in the wild type strain, This order of fluorescence intensity was fully consistent with a previously reported minimum inhibitory concentration of antibiotics such as tetracycline, chloramphenicol, and fluoroquinolones, Ethidium bromide accumulation in all the Mex mutants proceeded at about 5 times faster than the rate in the wild type cells, This result is in accord with the minimum inhibitory concentration of beta-lactam antibiotics. These results suggest that the fluorescence probes could be successfully used in real time monitoring of the function of the drug extrusion machinery in Gram-negative bacteria, The downhill extrusion kinetics of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, which orients perpendicular to the inner leaflet of the cytoplasmic membrane, from preloaded cells lacking the extrusion pump was preceded by a slow increase in fluorescence intensity, whereas the wild type cell immediately released the dye, This observation was explained by a slow trans-cytoplasmic membrane crossing of intracellular dye in the mutants, These results reflected higher accumulation of the probe in the cytoplasmic membrane in the mutants and strengthened the hypothesis that extrusion of hydrophobic substrate mediated by MexA-MexB-OprM mainly takes place from the interior of the cytoplasmic membrane.