1993 年度実績報告書

薬剤耐性に係わる細胞膜の機能及びそれと連係的に作用する因子の総合的研究

研究課題

研究課題/領域番号	05304028
研究機関	東海大学
研究代表者	中江太治東海大学, 医学部, 教授 (50102851)
研究分担者	河野恵東京薬科大学, 教授 (10057292) 平松啓一順天堂大学, 医学部, 教授 (10173262) 山口明人千葉大学, 薬学部, 助教授 (60114336) 井上松久北里大学, 医学部, 教授 (10008336) 伊豫部志津子群馬大学, 医学部, 助教授 (90008318)
キーワード	耐性 / 細菌 / 膜 / 抗生物質 / MRSA / 緑膿菌 / beta-ラクタマーゼ / クローニング
研究概要	細菌の薬剤耐性に係わる因子としては(i)薬剤修飾酵素の産生、(ii)薬剤透過性の低下及び(iii)標的の感受性低下などが主たる原因である。中でも細胞膜を介しての薬剤透過性の変化は他の二つの因子と連係的に働くことが多いので、本研究ではこれらの事を中心として共同研究態勢を組んだ。また研究の対称とした菌種は今日問題になっている緑膿菌、MRSA(ブドウ球菌)及び大腸菌などであった。 MRSAの研究に関してはMRSAのbeta-ラクタム耐性を決定するPBP2'をコードする遺伝子がどのようにしてブドウ球菌に押入されたのかの研究を行った。そのためにMRSAに特異的なDNAをクローン化して調べたところmecAに隣接してmecR1及びmecZの二つの調節遺伝子が存在する、とがわかった。これはLactobacillusのxyllと相同性を示した。同様にMRSAの消毒耐性のメカニズムを明らかとするため、これに係わる遺伝子をクローン化し発現させたところ、消毒剤耐性に係わる蛋白質を同定することができた。この結果消毒剤のこの蛋白質との親和性などを明らかにすることができた。MRSAの産生するロイコシジン及びgamma-ヘモリジンは標的細胞を障害するが、これら独立の毒素はサブユニットの一つを共有しているという興味ある結果を得た。緑膿菌はイミペネムに耐性化すると外膜蛋白質OprD2が欠損する。この外膜蛋白の欠損と耐性の機序をクローン化したOprD2遺伝子の変異を用いて検討したところ、耐性化と同時にOprD2が欠損するような変異菌ではOprD2遺伝子の一部に欠失変異が存在することが明らかとなった。また緑膿菌が多剤耐性化するときに発現されるOprM遺伝子及びその近傍の遺伝子をクローン化した。OprMの耐性に果たす役割は未解決である。緑膿菌のプラスミド上で発現するイミペネム/セフタジダイムを加水分解するbeta-lactamaseの遺伝子を含む領域をクローン化し、酵素の発現をみた。このbeta-ラクタマーゼはペニシリン類、第3世代セフエム、カルバペネム類、ペネム類など巾広いスペクトラムを示した。一方大腸菌におけるプラスミド性テトラサイクリン耐性は細胞内からテトラサイクリンの排出によることが示されている。そこでこの排出に係わる蛋白質と他の薬剤排出に係わる蛋白質のアミノ酸残基の基質特異性を示す部分の共通性を検索した。その結果膜貫通部分に共通性が見いだされた。この部分に突然変異を導入すると排出蛋白の基質特異性及び排出効率は大きく影響された。これらの研究打ち合わせ、研究発表会等を兼ねた班会議を一回行った。

研究成果
(32件)

すべてその他

すべて文献書誌 (32件)

[文献書誌] Hajime Ikigai and Taiji Nakae: "Bactericidal catechins damage the lipid bilayer" Biochimica Biophysica Acta. 1147. 132-136 (1993)
[文献書誌] Eisaku Yoshihara and Taiji Nakae: "Galcium ion-mediated opening of the channel Gate in the Pseudomonas aeruginosa porin" Biochemical Biophysical Research Commun. 194. 1460-1465 (1993)
[文献書誌] Hiroshi yoneyama and Taiji Nakae: "Mechanism of efficient elimination of ProteinD2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa" 37. 2385-2390 (1993)
[文献書誌] Junko Ishii and Taiji Nakae: "Lipopolysaccharide promoted opening of the porin channel" 3. 251-255 (1993)
[文献書誌] Shizuko Iyobe and H,Hashimoto: "Characteization of newly isolated plasmids from Actinobacillus pleuropneumoniae" Am.J.Vet.Res.54. 701-708 (1993)
[文献書誌] K,Hirai and Shizuko Iyobe: "Quinolone resistance in Pseudomonas aeruginosa Molecular Biology of DNA Topoisomerases and its Application to Chemoterapy" CRP Press. 157-165 (1993)
[文献書誌] Iyobe,S and Hashimoto,H: "Developments of low level norfloxacin resistance and its prevention in Pseudomonas aeruginosa" Recent Advances in Chemotherapy. 704-705 (1992)
[文献書誌] Iyobe,S and Hashimoto,H: "Occurrence of the nfxB type mutation in clinical isolates of Pseudomonas aeruginosa" Antimicrob.Agents.Chemother.36. 2562-2565 (1992)
[文献書誌] Suzuki,E and Hiramatsu,K: "Discribution of the novel genes,mecRI and mecl,in clinical strains of methicillin-resistant staphylococci" Antimicrob.Agents.Chemother.37. 1219-1226 (1993)
[文献書誌] Hiramatsu,K and Yokota,T: "Mutations in the rtbT gene are responsible for the Ogawa to lnaba serotype conversion in Vibrio choleree Ol" Microb.& lmmun.37(4). 281-288 (1993)
[文献書誌] Wada,A and Hiramatsu,K: "Molecular cloning and mapping of 16S-23S ribosomal RNA gene complexes in Staphylococcus aureus." J.Bacteriol.(in press).
[文献書誌] Hori,S and Hiramatsu,K: "Sequenrtial acquisition of newquinolone resistance by methicillin-resistant and Staphylococcus aureus." Antimicrob.Agents.Chemother.(in press).
[文献書誌] Yoshida,T and Hiramatsu,K: "Potent in Vitro bactericidal activity of polymyxin B against methicillin-resistant Staphylococcus aureus" Microbiol.lmmunol. (in press).
[文献書誌] Ito,T.Hiramatsu,K and Yokota,T: "ldentification of a novel sugar,2-0-methyl-4-amino-4,6-dideoxy mannose in the lipopolysaccharide of V.choleree 01 serotype Ogawa" Carbohydrate Research.(in press).
[文献書誌] Yasmin Abu Hanifah and Hiramatsu,K: "Pulsed-field gel electrophoresis(PFGE)of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections" MalaysianJ.Pathology.(in press).
[文献書誌] Sasatsu,M and Kono,M: "Detection of plasmid DNA in Erysipelothrix rhusiopathie isolated from pigs with chronic swine erysipelas" J.Vat.Med.Sci. 55(2). 349-350 (1993)
[文献書誌] Sasatsu,M and Kono,M: "Survival ability of clinical isolates of methicillin-resistant Staphylococcus aureus" Microbios.75. 17-21 (1993)
[文献書誌] Sasatsu,M and Kono,M: "Substrates and inhibitors of antiseptics resistance in Staphylococcus aureus" Biol.Pharm.Bull.(in press).
[文献書誌] Sasatsu,M and Kono,M: "Evaluation of antiseptics by the modified phenol coefficient method:sensitivity of methicillin-resistant Staphylococcus aureusto antiseptics." Biol.Pharm.Bull.(in press).
[文献書誌] Sasatsu,M and Kono,M: "High-level resistance to ethidium bronide and antiseptics in Staphylococcus aureus" FEMS Microbiol.Lett.93. 109-114 (1993)
[文献書誌] Sasatsu,M and Kono,M: "Triclosan-resistant Staphylococcus aureus" Lancet.341. (1993)
[文献書誌] Yamaguchi,A and Sawai,T: "Aspartic acid-66 is the only essential negatively chargedresidue in the putative hydrophilic loop region of the metal-tetracycline/H^+antiporter encoded by transposon Tn10 of Escherichia coli." Biochemistry.31. 8344-8348 (1992)
[文献書誌] Yamaguchi,A and Sawai,T: "Serine resicues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracvcline/H^+antiporter of Eschrichia coli." FEBS Letters. 307. 229-232 (1992)
[文献書誌] Yamaguchi,A and Sawai,T: "Metal-tetracycline/H^+antiporter of Escherichia coliencoded by transposon Tn10;The structural resemblance and functional difference in the role of the duplicated sequence motif between hydrophobic segments 2 and 3 and segments8and9" J.Biol.Chem.268. 6496-6504 (1993)
[文献書誌] Yamaguchi,A and Sawai,T: "Role of the conserved quartets of residues lacated in the N-and C-terminal halves of the transposon Tn10-encoded metal-tetracycline/H^+antiporter of Escherichia coli" Biochemistry. 32. 5698-5704 (1993)
[文献書誌] Yamaguchi,A and Sawai,T: "Effectsof sulthydry reagents on the Cys-65 mutant of the transposon Tn10encoded metal-tetracycline/H^+antiporter of Escherichia coli" FEBS.Letters. 322. 201-204 (1993)
[文献書誌] Yamaguchi,A and Sawai,T: "Effects sulthdry reagents on the Cry-65 mutant of the transposon Tn10encoded metal-tetracycline/H^+antiporter of Escherichia coli" FEBS.Letters.322. 201-204 (1993)
[文献書誌] Yamaguchi,A and Sawai,T: "Second-site mutation of Ala-220 toGlu or Asp suppresses the mutation of Asp285 to Asn in the transposon Tn10-encoded metal-tetracycline/H^+antiporter of Escherichia coli" J.Biol.Chem.(in press).
[文献書誌] Kamio,Y and Izaki,K: "The two Staphylococcal bi-component toxins,leukocidin and gamma-hemolysin,share one component in common" FEBS.lett.321. 15-18 (1993)
[文献書誌] Izaki,K and Kamio,Y: "Gamma-hemolysin genes in the same family with lukF and lukS genes in methicillin resistant Staphylococcus aureus" Biosci.Biotech.Biochem.57. 1234-1237 (1993)
[文献書誌] Izaki,K and Kamio,Y: "TheC-terminal region of S component of Staphylococcal leukocidin is essential for the biological activity of the toxin." FEBS.lett.329. 219-222 (1993)
[文献書誌] Nariya,H and Kamio,Y: "improved method for purification of leukocidin and gamma-hemolysin-components from Staphylococcus aureus" Biosci.Biotech.Biochem. (in press).

1993 年度 実績報告書

薬剤耐性に係わる細胞膜の機能及びそれと連係的に作用する因子の総合的研究

研究代表者

中江 太治 東海大学, 医学部, 教授 (50102851)

研究成果

[文献書誌] Hajime Ikigai and Taiji Nakae: "Bactericidal catechins damage the lipid bilayer" Biochimica Biophysica Acta. 1147. 132-136 (1993)

[文献書誌] Eisaku Yoshihara and Taiji Nakae: "Galcium ion-mediated opening of the channel Gate in the Pseudomonas aeruginosa porin" Biochemical Biophysical Research Commun. 194. 1460-1465 (1993)

[文献書誌] Hiroshi yoneyama and Taiji Nakae: "Mechanism of efficient elimination of ProteinD2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa" 37. 2385-2390 (1993)

[文献書誌] Junko Ishii and Taiji Nakae: "Lipopolysaccharide promoted opening of the porin channel" 3. 251-255 (1993)

[文献書誌] Shizuko Iyobe and H,Hashimoto: "Characteization of newly isolated plasmids from Actinobacillus pleuropneumoniae" Am.J.Vet.Res.54. 701-708 (1993)

[文献書誌] K,Hirai and Shizuko Iyobe: "Quinolone resistance in Pseudomonas aeruginosa Molecular Biology of DNA Topoisomerases and its Application to Chemoterapy" CRP Press. 157-165 (1993)

[文献書誌] Iyobe,S and Hashimoto,H: "Developments of low level norfloxacin resistance and its prevention in Pseudomonas aeruginosa" Recent Advances in Chemotherapy. 704-705 (1992)

[文献書誌] Iyobe,S and Hashimoto,H: "Occurrence of the nfxB type mutation in clinical isolates of Pseudomonas aeruginosa" Antimicrob.Agents.Chemother.36. 2562-2565 (1992)

[文献書誌] Suzuki,E and Hiramatsu,K: "Discribution of the novel genes,mecRI and mecl,in clinical strains of methicillin-resistant staphylococci" Antimicrob.Agents.Chemother.37. 1219-1226 (1993)

[文献書誌] Hiramatsu,K and Yokota,T: "Mutations in the rtbT gene are responsible for the Ogawa to lnaba serotype conversion in Vibrio choleree Ol" Microb.& lmmun.37(4). 281-288 (1993)

[文献書誌] Wada,A and Hiramatsu,K: "Molecular cloning and mapping of 16S-23S ribosomal RNA gene complexes in Staphylococcus aureus." J.Bacteriol.(in press).

[文献書誌] Hori,S and Hiramatsu,K: "Sequenrtial acquisition of newquinolone resistance by methicillin-resistant and Staphylococcus aureus." Antimicrob.Agents.Chemother.(in press).

[文献書誌] Yoshida,T and Hiramatsu,K: "Potent in Vitro bactericidal activity of polymyxin B against methicillin-resistant Staphylococcus aureus" Microbiol.lmmunol. (in press).

[文献書誌] Ito,T.Hiramatsu,K and Yokota,T: "ldentification of a novel sugar,2-0-methyl-4-amino-4,6-dideoxy mannose in the lipopolysaccharide of V.choleree 01 serotype Ogawa" Carbohydrate Research.(in press).

[文献書誌] Yasmin Abu Hanifah and Hiramatsu,K: "Pulsed-field gel electrophoresis(PFGE)of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections" MalaysianJ.Pathology.(in press).

[文献書誌] Sasatsu,M and Kono,M: "Detection of plasmid DNA in Erysipelothrix rhusiopathie isolated from pigs with chronic swine erysipelas" J.Vat.Med.Sci. 55(2). 349-350 (1993)

[文献書誌] Sasatsu,M and Kono,M: "Survival ability of clinical isolates of methicillin-resistant Staphylococcus aureus" Microbios.75. 17-21 (1993)

[文献書誌] Sasatsu,M and Kono,M: "Substrates and inhibitors of antiseptics resistance in Staphylococcus aureus" Biol.Pharm.Bull.(in press).

[文献書誌] Sasatsu,M and Kono,M: "Evaluation of antiseptics by the modified phenol coefficient method:sensitivity of methicillin-resistant Staphylococcus aureusto antiseptics." Biol.Pharm.Bull.(in press).

[文献書誌] Sasatsu,M and Kono,M: "High-level resistance to ethidium bronide and antiseptics in Staphylococcus aureus" FEMS Microbiol.Lett.93. 109-114 (1993)

[文献書誌] Sasatsu,M and Kono,M: "Triclosan-resistant Staphylococcus aureus" Lancet.341. (1993)

[文献書誌] Yamaguchi,A and Sawai,T: "Aspartic acid-66 is the only essential negatively chargedresidue in the putative hydrophilic loop region of the metal-tetracycline/H^+antiporter encoded by transposon Tn10 of Escherichia coli." Biochemistry.31. 8344-8348 (1992)

[文献書誌] Yamaguchi,A and Sawai,T: "Serine resicues responsible for tetracycline transport are on a vertical stripe including Asp-84 on one side of transmembrane helix 3 in transposon Tn10-encoded tetracvcline/H^+antiporter of Eschrichia coli." FEBS Letters. 307. 229-232 (1992)

[文献書誌] Yamaguchi,A and Sawai,T: "Role of the conserved quartets of residues lacated in the N-and C-terminal halves of the transposon Tn10-encoded metal-tetracycline/H^+antiporter of Escherichia coli" Biochemistry. 32. 5698-5704 (1993)

[文献書誌] Yamaguchi,A and Sawai,T: "Effectsof sulthydry reagents on the Cys-65 mutant of the transposon Tn10encoded metal-tetracycline/H^+antiporter of Escherichia coli" FEBS.Letters. 322. 201-204 (1993)

[文献書誌] Yamaguchi,A and Sawai,T: "Effects sulthdry reagents on the Cry-65 mutant of the transposon Tn10encoded metal-tetracycline/H^+antiporter of Escherichia coli" FEBS.Letters.322. 201-204 (1993)

[文献書誌] Yamaguchi,A and Sawai,T: "Second-site mutation of Ala-220 toGlu or Asp suppresses the mutation of Asp285 to Asn in the transposon Tn10-encoded metal-tetracycline/H^+antiporter of Escherichia coli" J.Biol.Chem.(in press).

[文献書誌] Kamio,Y and Izaki,K: "The two Staphylococcal bi-component toxins,leukocidin and gamma-hemolysin,share one component in common" FEBS.lett.321. 15-18 (1993)

[文献書誌] Izaki,K and Kamio,Y: "Gamma-hemolysin genes in the same family with lukF and lukS genes in methicillin resistant Staphylococcus aureus" Biosci.Biotech.Biochem.57. 1234-1237 (1993)

[文献書誌] Izaki,K and Kamio,Y: "TheC-terminal region of S component of Staphylococcal leukocidin is essential for the biological activity of the toxin." FEBS.lett.329. 219-222 (1993)

[文献書誌] Nariya,H and Kamio,Y: "improved method for purification of leukocidin and gamma-hemolysin-components from Staphylococcus aureus" Biosci.Biotech.Biochem. (in press).

1993 年度実績報告書

中江太治東海大学, 医学部, 教授 (50102851)