| Co-Investigator(Kenkyū-buntansha) |
TSUDA Masataka Tohoku University, Research Center of Genetic Ecology, Professor, 遺伝生態研究センター, 教授 (90172022)
NISHINO Takeshi Kyoto Pharmaceutical University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (50097838)
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| Research Abstract |
Our project has been focused on researches on molecular mechanisms of multicomponent efflux systems, which involved to broad-spectrum cross-resistance of Pseudomonas aeruginosa and obtained the following results. 1) A new efflux system MexX-MexY functions under collaboration with OprM, which expresses in the wild-type strains, such as PAO1. 2) Several antimicrobial agents such as erythromycin, tetracycline and gentamicin de-represses expression of MexX-MexY, which is suppressed in the wild-type cells. 3) All of these efflux systems extrude a wide variety of antimicrobial agent groups such as quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (except carbenicillin and sulbenicillin), most cephems (except cefsulodin and ceftazidime), meropenem and S-4661, but not imipenem and polymyxin B.Nonetheless, the respective substrate specificities to some agents are distinguishable. 4) The RND family proteins are assumed to function as an exporting component, alt
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hough there is little experimental evidence to support this assumption. To verify the assumption, based on the MexC-MexD-OprJ efflux system of Pseudomonas aeruginosa, we constructed eight chimeric efflux systems, in which MexD was exchanged for other RND proteins. RND proteins such as MexB and MexD are primary components of the efflux systems that function in the recognition of extrusion substrates. 5) To investigate the topology of MexD more thoroughly, 25 MexD-PhoA (alkaline phosphatase) and 18 MexD-Bla (β-lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N and C termini are located in the cytoplasm and the predicted orientation is consistent with the "positive-inside rule". This topological model can be applied to other RND proteins. Furthermore, analysis of mutated MexD from the clinical isolates showed that the two periplasmic loops function to recognition of substrates. Less
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