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

机会致病菌铜绿假单胞菌对多种结构无关的抗生素表现出高水平的多重内在耐药。众所周知，铜绿假单胞菌的这种多药耐药表型是由于存在广泛特异性的药物外排系统和低通透性的外膜。迄今为止，已在铜绿假单胞菌中确定了7个RND多药物外排泵，并显示它们由3个蛋白质亚基组成。MexAB-OprM是主要的组成表达的多药物外排泵，该泵的三个亚基被证明是其外排功能不可或缺的。然而，这些亚基是如何组合形成泵复合物的尚不清楚，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测了MexAB-OprM的亚基相互作用如下。当研究两个亚基之间的相互作用时，一个亚基在其C-ter上被his标记，更容易与Ni-NTA树脂结合。随后，在该柱上应用了另一个亚基。如果这些蛋白相互作用，第二个亚基可以通过蛋白-蛋白相互作用结合在柱上。通过这种方法，我们测量了(1)His-tagged的MexA和OprM， (2) His-tagged的MexB和MexA， (3) His-tagged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。综上所述，这表明内膜MexB直接与外膜OprM通道相互作用，允许药物通过绕过外周质的途径，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，该方法不适用于筛选系统中抑制泵亚基相互作用的化合物。因此，我们尝试开发利用荧光共振能量转移（FRET）的方法。该方法要求蛋白分别用FRET供体和受体荧光染料修饰。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。当抑制剂被加入到混合物中时，FRET被认为会消失，因为两种蛋白质被分离了。目前我们正在尝试制备分别用FRET给体和受体染料修饰的MexA和OprM。少

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