Structure and function of drug- and ion-extrusion systems in bacteria

从结构和功能方面对副溶血性弧菌、铜绿假单胞菌、金黄色葡萄球菌和枯草芽孢杆菌的药物挤出系统和离子挤出系统进行了研究。在药物挤出系统方面，我们发现了几个新的药物外排系统，分别是副溶血性弧菌的NorM、铜绿假单胞菌的MexXY和枯草芽孢杆菌的EbrAB。钠离子刺激药物（乙锭）经NorM外排。在预先加载乙锭的能量饥渴细胞中施加向内定向Na+梯度会引起乙锭的外排。在预先加载Na+的能量匮乏细胞中施加向内定向的乙锭梯度可引起Na+的外排。这些结果清楚地表明，药物从细胞中挤出以换取Na+，即NorM是药物/Na+的反向转运体。这是生物学界第一个药物/Na+逆向转运体的例子。从数据库中发现的序列相似性来看，似乎在其他细菌中也存在一些药物/Na+反转运蛋白。发现MexXY为三组分型多药外排泵。该系统参与铜绿假单胞菌对红霉素、氨基糖苷类和β-内酰胺类的耐药。另一种外排系统是离子外排系统，我们发现了几种Na+外排系统并进行了分析，分别是金黄色葡萄球菌的Mnh、副溶血性弧菌的NhaB和枯草芽孢杆菌的NhaG。这些体系挤出Na+以换取H+。Mnh系统是一种新型的Na+/H+反转运体，多亚基型反转运体。我们还用膜片钳法分析了大肠杆菌细胞的离子外排。这种方法可以直接测量细菌细胞中的离子通量。

We investigated drug extrusion system and ion extrusion systems in Vibrio parahaemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis from structural and functional points of view. Regarding drug extrusion systems, we found several new drug efflux systems, NorM from V. parahaemolyticus, MexXY from P. aeruginosa and EbrAB from B. subtilis. Drug (ethidium) efflux via the NorM was stimulated by Na+. Imposition of inwardly directed Na+ gradient in energy starved cells that were preloaded with ethidium elicited efflux of ethidium. Imposition of inwardly directed ethidium gradient in energy starved cells that were preloaded with Na+ elicited efflux of Na+. These results clearly indicate that drug is extruded from cells in exchange for Na+, namely the NorM is a drug/Na+ antiporter. This is the first example of drug/Na+ antiporter in biological world. Judging from sequence similarity found in databases, it seems that there are some drug/Na+ antiporters in some other bacteria. The MexXY was found to be three-components type multidrug efflux pump. This system is involved in resistance of P. aeruginosa against erythromycin, aminoglycosides and β-lactams. Regarding another efflux systems, ion efflux systems, we found several Na+ efflux systems and analyzed them, Mnh from S. aureus, NhaB from V. parahaemolyticus and NhaG from B. subtilis. These systems extrude Na+ in exchange for H+. The Mnh system is a novel type Na+/H+ antiporter, multisubunit type antiporter. We also analyzed ion efflux from E. coli cells using patch clamp method. This method enabled direct measurement of ion flux in bacterial cells.