The elucidation of the molecular assembly mechanism of the multidrug efflux pump and the development of the screening system for pump inhibitors

多药外排泵分子组装机制的阐明及泵抑制剂筛选体系的开发

• 批准号: 14570245
• 负责人: YOSHIHARA Eisaku
• 金额: $ 1.86万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570245/
• 关键词: Pseudomonas aeruginosa; multidrug resistance; drug efflux pump; MexAB-OprM pump; subunit; protein-protein interaction; inhibitors; FRET; 院内感染; 多剤排出ポンプ; MexAB-OprM; ヒスチジンタグ; アフィニティカラム; HlyBD-TolC

The opportunistic pathogen Pseudomonas aeruginsa displays high-level multiple intrinsic resistance to a variety of structurally unrelated antibiotics. It is known that this multidrug resistance phenotype of P.aeruginosa results from the presence of broad-specificity drug efflux-systems and low-permeability outer membrane. To date, seven RND multidrug efflux pumps have been identified in P.aeruginosa and shown to be composed of three protein subunits. MexAB-OprM is the major and constitutively expressed multidrug efflux pump and three subunits of this pump are demonstrated to be indispensable for its efflux function. However, it is unclear how these subunits assemble to form the pump complex, which is the essential issue to elucidate the molecular functional mechanism of the efflux pump. In order to answer the problem, we examined the subunit interactions of MexAB-OprM in vitro system as follows. When examine the interactions between two subunits, one subunit was His-tagged at its C-ter … More minal to bind to the Ni-NTA resin. Subsequently another subunit was applied on this column. If these proteins interact each other, second subunit could bind on the column through the protein-protein interaction. By using this method we measured the interaction between (1) His-tagged MexA and OprM, (2) His-tagged MexB and MexA and (3) His-tagged MexB and OprM. The results clearly showed that these subunits interact in all cases. All together it was suggested that the inner membrane MexB directly interacts with the outer membrane OprM channel allowing drugs to pass the route bypassing the periplasm, and MexA stabilizes the pump complex through the protein-protein interactions. On the other hand this method is not suitable for the screening system to search the compounds which inhibit the pump subunit interactions. Therefore we tried to develop the method utilizing the fluorescence resonance energy transfer (FRET). This method requires the proteins modified with FRET donor and acceptor fluorescence dye, respectively. If these proteins are able to interact each other, FRET is expected to occur because two dyes are in the proximity. When the inhibitors are added to the mixture, FRET is though to disappear because two proteins are separated. At present we are trying to prepare the MexA and OprM modified with FRET donor and acceptor dyes, resectively. Less

条件致病菌铜绿假单胞菌对多种结构无关的抗生素表现出高度的多重内在耐药性。已知铜绿假单胞菌的这种多药耐药表型是由广泛特异性药物外排系统和低渗透性外膜的存在引起的。迄今为止，已经在铜绿假单胞菌中鉴定了七种RND多药外排泵，并且显示其由三种蛋白质亚基组成。MexAB-OprM是主要的组成型多药外排泵，该泵的三个亚基被证明是其外排功能不可缺少的。然而，这些亚基如何组装形成泵复合物尚不清楚，这是阐明外排泵分子功能机制的关键问题。为了回答这个问题，我们在体外系统中检查了MexAB-OprM的亚基相互作用，如下所示。当检测两个亚基之间的相互作用时，一个亚基在其C端被His标记， ...更多信息 以结合到Ni-NTA树脂。随后，将另一个亚单元施加在该柱上。如果这些蛋白质相互作用，第二亚基可以通过蛋白质-蛋白质相互作用结合在柱上。通过使用该方法，我们测量了（1）His-标记的MexA和OprM，（2）His-标记的MexB和MexA以及（3）His-标记的MexB和OprM之间的相互作用。结果清楚地表明，这些亚基在所有情况下都相互作用。总之，表明内膜MexB直接与外膜OprM通道相互作用，允许药物绕过周质通过该途径，并且MexA通过蛋白质-蛋白质相互作用稳定泵复合物。另一方面，这种方法不适合于筛选系统来寻找抑制泵亚基相互作用的化合物。因此，我们试图开发利用荧光共振能量转移（FRET）的方法。该方法需要分别用FRET供体和受体荧光染料修饰的蛋白质。如果这些蛋白质能够相互作用，FRET预计会发生，因为两种染料在附近。当抑制剂加入到混合物中时，FRET会消失，因为两种蛋白质被分离。目前，我们正在尝试分别用FRET供体和受体染料修饰MexA和OprM。少

项目成果

Maseda, H.: "A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa"Mol.Microbiol.. 46. 677-686 (2002)

Maseda, H.：“铜绿假单胞菌中多组分异生物质外排泵的新型组装工艺”Mol.Microbiol.. 46. 677-686 (2002)

Saito, K.: "Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression"J.Bacteriol.. 185. 6195-6198 (2003)

Saito, K.：“影响 mexR-mexA-mexB-oprM 操纵子表达的 MexR 阻遏物 DNA 结合活性的突变”J. Bacteriol.. 185. 6195-6198 (2003)

Yoshihara, E.: "The outer membrane component of the multidrug efflux pump from Pseudomonas aruginosa may be a gated channel"Eur.J.Biochem.. 267. 4738-4745 (2002)

Yoshihara, E.：“来自铜绿假单胞菌的多药外排泵的外膜成分可能是门控通道”Eur.J.Biochem.. 267. 4738-4745 (2002)

Narita, S.: "Linkage of the efflux-pump expression level with substrate extrusion rate in the MeAB-OprM efflux pump of Pseudomonas aeruginosa"Biochem.Biophys.Res.Conimun.. 308. 922-926 (2003)

Narita，S.：“铜绿假单胞菌 MeAB-OprM 外排泵中外排泵表达水平与底物挤出速率的关联”Biochem.Biophys.Res.Conimun.. 308. 922-926 (2003)

Maseda: "A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa"Molecular Microbiology. 46. 677-686 (2002)

Maseda：“铜绿假单胞菌中多组分异生物质外排泵的新型组装工艺”分子微生物学。