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

细菌中药物和离子挤出系统的结构和功能

• 批准号: 09672230
• 负责人: TSUCHIYA Tomofusa
• 金额: $ 1.73万
• 依托单位: OKAYAMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672230/
• 关键词: Drug extrusion system; Ion transport; Pseudomonas aeruginosa; Vibrio parahaemolyticus; Patch clamp; 黄色ブドウ球菌

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.

我们从结构和功能的角度研究了副溶血性弧菌、铜绿假单胞菌、金黄色葡萄球菌和枯草杆菌的药物排泄系统和离子排泄系统。在药物外排系统方面，我们发现了几个新的药物外排系统，包括来自副溶血弧菌的Norm，来自铜绿假单胞菌的Mexxy和来自枯草杆菌的EbrAB。钠离子刺激药物(乙锭)经正常外排。在预加乙锭的能量饥饿细胞中施加内向Na+梯度可引起乙锭外流。在预加Na+的能量饥饿细胞中施加内向乙锭梯度可引起Na+外流。这些结果清楚地表明药物被挤出细胞以换取Na+，即正常情况下是药物/Na+逆向转运蛋白。这是生物界首次发现药物/Na+逆向转运蛋白。从数据库中发现的序列相似性来看，似乎在其他一些细菌中存在一些药物/Na+逆向转运蛋白。Mexxy为三组分型多药外排泵。该系统参与了铜绿假单胞菌对红霉素、氨基糖苷类和β-内酰胺类抗生素的耐药。对于另一种外排系统，离子外排系统，我们发现了几个Na+外排系统，并对它们进行了分析，包括来自金黄色葡萄球菌的MNH，来自副溶血弧菌的NhaB和来自枯草杆菌的NhaG。这些体系排出Na+以换取H+。MNH系统是一种新型的Na+/H+逆向转运蛋白，多亚单位类型的逆向转运蛋白。我们还用膜片钳方法分析了大肠杆菌细胞的离子外流。这种方法可以直接测量细菌细胞中的离子通量。

项目成果

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Teruo Kuroda 等人：“大肠杆菌细胞质膜离子泵的膜片钳研究：由翻转细胞质膜组成的巨型液泡样结构的形成、制备和利用”J.

Teuo Kuroda, et al.: "Mutational analysis of amiloride sensitivity in NhaA Na+/H+ antiporter from Vibrio parahaemolyticus"J. Bacteriol.. 179. 7600-7602 (1997)

Teuo Kuroda 等人：“副溶血弧菌 NhaA Na /H 反向转运蛋白中阿米洛利敏感性的突变分析”J。

Rafiquel I.Sarker: "Primary structure and properties of the Na+/glucose symporter (SglS) of Vibrio parahaemolyticus." Journal of Bacteriology. 179. 1805-1808 (1997)

Rafiquel I.Sarker：“副溶血弧菌钠/葡萄糖同向转运体 (SglS) 的主要结构和特性。”

Hiroki Inoue: "Expression of functional Na+/H+ antiporters of helicobacter pylori in antiporter deficient Escherichia coli mutants"FEBS Lett.. 443. 11-16 (1999)

Hiroki Inoue：“幽门螺杆菌功能性 Na /H 逆向转运蛋白在反向转运蛋白缺陷型大肠杆菌突变体中的表达”FEBS Lett.. 443. 11-16 (1999)

Kazuyo Kodama, et al.: "Preparation and characterization of everted membrane vesicles from cells of Saphylococcus aureus"Biol. Pharm. Bull.. 21. 5-9 (1998)

Kazuyo Kodama 等人：“金黄色葡萄球菌细胞外翻膜囊泡的制备和表征”Biol。