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

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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