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

Research Project

Project/Area Number 14570245
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Bacteriology (including Mycology)
Research InstitutionTokai University

Principal Investigator

YOSHIHARA Eisaku  Tokai University, School of Medicine, Associate Professor, 医学部, 助教授 (70167063)

Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,700,000 (Direct Cost: ¥1,700,000)
KeywordsPseudomonas 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
Research Abstract

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

Report

(3 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Yoshihara: "The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel"Eur.J.Biochem.. 269. 4738-4745 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Maseda: "A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa"Molecular Microbiology. 46. 677-686 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Narita: "Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa"Biochem.Biophys.Res.Commun.. 308. 922-926 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Saito: "Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression"J.Bacteriol.. 185. 6195-6198 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Maseda, H.: "A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa"Mol.Microbiol.. 46. 677-686 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Saito, K.: "Mutations affecting DNA-binding activity of the MexR repressor of mexR-mexA-mexB-oprM operon expression"J.Bacteriol.. 185. 6195-6198 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Saito: "Mutations affecting DNA-binding activity of the MexR represser of mexR-mexA-mexB-oprM operon expression"J.Bacteriol.. 185・20. 6195-6198 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Narita: "Linkage of the efflux-pump expression level with substrate extrusion rate in the MexAB-OprM efflux pump of Pseudomonas aeruginosa"Biochem.Biophys.Res.Commun.. 308. 922-926 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Yoshihara: "The outer membrane component of the multidrug efflux pump from Pseudomonas aeruginosa may be a gated channel"Eur. J. Biochem.. 269. 4738-4745 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Maseda: "A novel assembly process of the multicomponent xenobiotic efflux pump in Pseudomonas aeruginosa"Molecular Microbiology. 46(3). 677-686 (2002)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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