1991 Fiscal Year Final Research Report Summary
Role of the outer membrane permeability in the antibiotic resistance in Pseudomonas aerugionsa
| Project/Area Number |
02454179
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | Tokai University |
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Principal Investigator |
NAKAE Taiji Tokai University School of Medicine, Professor, 医学部, 教授 (50102851)
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| Co-Investigator(Kenkyū-buntansha) |
YONEYAMA Hiroshi Tokai University School of Medicine, Instructor, 医学部, 助手 (10220774)
SATAKE Sachiko Tokai University School of Medicine, Researcher, 医学部, 研究員
ISHII Junko Tokai University School of Medicine, Instructor, 医学部, 助手 (20212813)
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| Project Period (FY) |
1990 – 1991
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| Keywords | Outer membrane / Permeability / Liposome / Planar membrane / Drug resistance / Cloning / Membrane protein / Reconstitution |
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| Research Abstract |
Pseudomonas aeruginosa is a pathogen to the immunocompromised host. Infection of this bacteria is a problem, since the bacteria is highly resistant to a number of structurally unrelated antibiotics. This high antibiotic resistance is due to low outer membrane permeability. To elucidate the role of the outer membrane permeability to the antibiotic resistance. we investigated the permeability properties of the porins. Purified outer membrane porins, proteins C, D2 and E1 were reconstituted into a planar membrane of diphytanoyl phosphatidyl choline in 1 M NaCl solution and the conductivity across the membrane was determined. The single channel conductance of protein C and El in I M NaCl appeared to be about 120 pS and 240 pS, respectively. The single channel conductance of protein D2 showed two discrete channel sizes of 10-30 pS and about 300-500 pS. The channel sizes are far smaller than that of E. coli porins (1200-1400 pS).
Purified proteins C, D2 and El were reconstituted into the Iipo
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some membrane and the antibiotic permeability was determined. Among beta-lactam antibiotics, only carbapems, such as imipenem and meropenem showed efficient diffusion through the porin pores. Protein D2 showed higher efficiency in the imipenem permeability tha nother two porins. Other cephalosporins diffused through these porin pores little. Thus, role of protein D2 in the imipenem diffusion was clarified in vitro.
Imipenem diffusion via protein D2 in vitro was tested by clonig P. aeruginosa gene coded for protein D2. The cloned gene was expressed in the protein D2-deficient host and over produced protein D2. The Protein D2-deficient host fully restored protein D2 upon harboring the protein D2-cloned plasmid.
Thus, the role of protein D2 in the imipenem permeability was demonstrated in vitro.
These studies clearly demonstrated that the outer membrane pores of Pseudomonas aeruginosa are far smaller than that of E. coli. Protein D2 is only the porin practically functioning in the carbapenem diffusion and the role of other porins in the antibiotic diffusion seems to be a little. Less
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