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1999 Fiscal Year Final Research Report Summary

Structure and Function of the Antibiotic-Specific Channel

Research Project

Project/Area Number 10044320
Research Category

Grant-in-Aid for Scientific Research (B).

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionTokai University

Principal Investigator

NAKAE Taiji  Tokai University School of Medicine, Professor, 医学部, 教授 (50102851)

Co-Investigator(Kenkyū-buntansha) YONEYAMA Hiroshi  Tokai University School of Medicine, Assistant Professor, 医学部, 講師 (10220774)
YOSHIHARA Eisaku  Tokai University School of Medicine, Associate Professor, 医学部, 助教授 (70167063)
Project Period (FY) 1998 – 1999
KeywordsAntibiotics / Drug resistance / Efflux / Pump / Membrane protein / Crystal / X-ray diffraction / Infection
Research Abstract

Pseudomonas aeruginosa is a major pathogen in the hospital. Infection of this organism is a large problem, since this bacterium exhibits resistance to multiple species of antibiotics that is largely due to a tight outer membrane barrier and the expression of xenobiotic efflux pump. A major efflux pump in this organism is the MexAB-OprM pump. Aim of this study is to elucidate structure and function of the pump subunits. (I) The MexA subunit is an inner membrane associated protein assumed to link the MexB and OprM subunits. We tried to crystalize native from to MexA in the presence of surfactant, polyethyleneglycol 2000, LiCl and obtained 100 to 150 μmィイD13ィエD1 crystals. Preliminary X-ray diffraction analysis showed about 4 AィイD4゜ィエD4 resolution. Deacylated MexA removing fatty acid moiety by replacing N-terminal cysteine with other amino acid was also crystallized in the absence of surfactant and the current resolution is about 3.8 ィイD4゜ィエD4A. (ii) The OprM protein was purified by tagging poly-histidine extension at the carboxyl terminal end and purifying by Nickel-Sepharose column. We obtained about 100〜150 μmィイD13ィエD1 crystals in the presence of octylhydroxyethylsulfoxide, NaCl, polyethylene glycol 2000. Present resolutions is about 4.8 AィイD4゜ィエD4. Obviously more refined crystallization conditions has to be set up. (iii) The MexB structure has been studied by the gene-fusion of a reporter gene, phoA, into the C-terminal truncated MexB protein. Expression of such hybrid proteins revealed that the MexB protein bears 12 transmembrane segments leaving N- and C- termini in the cytoplasm. The MexB protein had two large periplasmic domain containing 311 and 314 amino acid residues. The MexB protein showed two-fold symmetry having highly conserved N- and C-termini halves. These properties are unique to the MexB protein.

  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Monika Germ: "Interplay between the efflux pump and the outer membrane permealibity barrier in fluorescent dye accumulation in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 261. 452-455 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hajime Ikigai: "Two forms of Vibrio chlerae O1 El Tor hemolysin derived from identical precursor protein"Biochem. Biophys. Acta. 1415. 297-305 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Taiji Nakae: "Resistance to beta-lectam antibiotics in Pseudomonas aeurinosa by interplay between the MexA, B-OprM efflux pump and beta-lactamase"Antimicrob. Agents Chemother. 43. 1301-1303 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Kojiro Saito: "NalB-type mutaitons causing the over expression of the MexAB-OprM efflux pump are located in the MexR gene of Pseudomonas aeruginosa chromosome"FEMS Microbiology Letters. 179. 67-72 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Lan Guan: "Membrane topology of the xenobiotic-exporting subunit, MexB of the MexA, B-OprM extrusion pump in Pseudomonas aeruginosa"J. Biol. Chem.. 274. 10517-10522 (1999)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Keiko Suzuki: "Flocculation and Membrane Binding of Outer Membrane Protein F, Porin, at Acidic pH"Jpn. J. Appl. Phys.. 37. 2074-2077 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Akira Nakajima: "Antibiotic stress induces a large amount of outer membrane protein in Pseudomomas aeruginosa"FEMS Microbiology Letters. 165. 261-265 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Eisaku Yoshihara: "Identification of the Catalytic Triad of the Protein D2 Protease in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 247. 142-145 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Keiko Suzuki: "Subunit Swapping in the Mex-Extrusion Pumps in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 244. 898-902 (1998)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Monika Germ, Eisaku Yoshihara, Hiroshi Yoneyama and Taiji Nakae: "Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 261. 452-455 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hajime Ikigai, Toshihisa Ono, Taiji Nakae, Hiaroshi Otsuru and Tadakatsu Shimamura: "Two forms of Vibrio cholerae 01 EI Tor hemolysin derived from identical precursor protein."Biochem. Biophys. Acta. 1415. 297-305 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Taiji Nakae, Akira Nakajima, Toshihisa Ono, Kohjiro Saito and Hiroshi Yoneyama: "Resistance to beta-lactam antibiotics in Pseudomonas aeruginosa by interplay between the MexA,B-OprM efflux pump and beta-lactamase"Antimicrob. Agents Chemother. 43. 1301-1303 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Kohjiro Saito, Hiroshi Yoneyama, Taiji Nakae: "NalB-type mutaitons causing the over expression of the MexAB-OprM efflux pump are located in the mexR gene of Pseudomonas aeruginosa chromosome"FEMS Microbiology Letters. 179. 67-72 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Lan Guan, Michael Ehrmann, Hiroshi Yoneyama and Taiji Nakae: "Membrane topology of the xenobiotic-exporting subunit, MexB of the MexA,B-OprM extrusion pump in Pseudomonas aeruginosa"J. Biol. Chem.. 274. 10517-10522 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Akira Nakajima, Mie Hoshikawa, Taiji Nakae: "Antibiotic stress induces a large amount of outer membrane protein in Pseudomonas aeruginosa"FEMS Microbiology Letters. 165. 261-265 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Eisaku Yoshihara, Hiroshi Yoneyama, Toshihisa Ono, Taiji Nakae: "Identification of the Catalytic Triad of the Protein D2 Protease in Pseudomonas aeruginosa"Biophys. Res. Commun.. 247. 142-145 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Keiko Suzuki, Taiji Nakae, Shigeki Mitaku: "Flocculation and Membrane Binding of Outer Membrane Protein F, Porin, at Acidic pH"Jpn. J. Appl. Phys. 37. 2074-2077 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hidroshi Yoneyama, Aydin Ocaktan, Naomasa Gotoh, Takeshi Nishino, and Taiji Nakae: "Subunit Swapping in the Mex-Extrusion Pumps in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 244. 898-902 (1998)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 2001-10-23  

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