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1999 Fiscal Year Final Research Report Summary

The xenobiotic efflux pumps and multiantibiotic resistance in Pseudomonas aeruginosa

Research Project

Project/Area Number 09470079
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Bacteriology (including Mycology)
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) 1997 – 1999
KeywordsAntibiotic resistance / Pump / Membrane protein / Membrane topology / Membrane / Transport / Pseudomonas / Efflux
Research Abstract

Pseudomonas aeruginosa is a major pathogen in the hospital that exhibits resistance to many structurally and functionally diverse antibiotics, This multidrug resistance is mainly attributable to a tight outer membrane permeability and expression of the xenobiotic efflux pumps. The MexAB-OprM efflux pump consists of three subunits such as inner membrane protein, MexB, inner membrane associated periplasmic protein, MexA and outer membrane protein, OprM, functioning as the energy dependent efflux pump, the membrane fusion protein and the xenobiotic exit channel, respectively, We studied structure and function of these pump subunit proteins. (I) To acess the role of the subunit proteins in the antibiotic export, we constructed by mean of gene replacement technique the mutant lacking either MexA, MexB, OprM or all combinations of these subunits. The antibiotic susceptibility test of these mutant revealed that the mutants lacking MexA or MexB are equally hypersusceptible to many antibiotics, … More whereas the mutants lacking the OprM subunit are more antibiotic hypersusceptible than the MexA or MixB mutants. (ii) To quantitate the pump function in real time, we developed the method to determine the fluorescent dye accumulation in stead of measuring the antibiotic susceptibility. The dyes accumulation in the cell was proportionally related with the antibiotic susceptibility of the cells. Thus, the measurement of fluorescent dye accumulation could be used as a pump function monitor. (iii) P. aeruginosa encodes MexCD-OprJ pump upon the regulator mutation, the nfxB gene. Since the amino acid sequence of the MexAB-OprM and MexCD-OprJ has high similarity, we tested a possible interchangeability of the pump subunits, expressing the hybrid pump. To do it, OprJ subunit, for instance, was expressed in the host lacking OprM subunit and producing the MexA and MexB subunits. Results of such study revealed that the outer membrane subunit could be substituted, but not the inner membrane subunit. Thus, it became clear that P. aeruginosa expressing more than two pump systems produces the hybrid pumps. (iv) The MexB subunit is assumed to be the energy dependent efflux pump crossing the cytoplasmic membrane several times. However, there is no experimental manifestation to this assumption. We studied the membrane topology of the MexB protein by the reporter gene (phoA ) fusion method to the various length of carboxyl terminal truncated mexB gene. Expression of such fusion genes showed that the alkaline phosphatase activity of the fusion protein, of which fusion-site is faced to the periplasm would be high. On one hand, the enzyme activity would be undetectably low in the hybrid protein, of which reporter gene is located in the cytoplasm. We constructed a total 28 hybrid proteins. The result showed that the MexB protein bears 12 transmembrane domains leaving the amino and carboxyl termini in the cytoplasm. Interestingly, the MexB protein had two large hydrophilic domains containing 311 and 314 amino acid residues. Thus, it was suggested that these two large domains may interact with the MexA and Oprm subunits. Less

  • Research Products

    (28 results)

All Other

All Publications (28 results)

  • [Publications] Monika Germ: "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(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] 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: "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] 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] Taiji Nakae: "Multiantibbiotic resistance caused by active drug extrusion in the hospital pathogen"J. Infect. Chemother.. 3. 173-183 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Takashi Kitahara: "Antibiotic diffusion pathways in the outer membrane of Peudomonas aer4g5n6sa"Biochem. Biophys. Res. Commun.. 238. 457-461 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Aydin Ocaktan: "Use of fluorescence probes to monitor function of the subunit proteins of the Mex-A-MexB-OprM drug extrusion machinery in Pseudomonas aeruginosa"J. Biol. Chem.. 272. 21964-21969 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Hiroshi Yoneyama: "The role of mex-gene products in antibiotic extrusin in Pseudomonas aeruginosa."Biochem. Biophys. Res. Commun.. 233. 611-618 (1997)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Taiji Nakae: "Multiantibiotic resistance caused by active drug extrusion in Pseudomonas aeruginosa and other gram-negative bacteria."Microbiologia. 13. 273-284 (1997)

    • 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 and 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
  • [Publications] Taiji Nakae, Eisaku Yoshihara, and Hiroshi Yoneyama: "Multiantibiotic resistance caused by active drug extrusion in the hospital pathogen"J. infect. Chemother.. 3. 173-183 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Takashi Kitahara, Hiroshi Yoneyama, and Taiji Nakae: "Antibiotic diffusion pathways in the outer membrane of Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 238. 457-461 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Aydin Ocaktan, Hiroshi Yoneyama, and Taiji Nakae: "Use of fluorescence probes to monitor function of the subunit proteins of the Mex-A-MexB-OprM drug extrusion machinery in Pseudolnonas aeruginosa"J. Biol. Chem.. 272. 21964-21969 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Hiroshi Yoneyama, Aydin Ocaktan, MasatakaTsuda,.M.,and Taiji Nakae: "The role of mex-gene products in antibiotic extrusion in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 233. 611-618 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Taiji Nakae: "Multiantibiotic resistance caused by active drug extrusion in Pseudomonas aeruginosa and other gram-negative bacteria."Microbiologia. 13. 273-284 (1997)

    • 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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