MOLECULAR AND GENETICAL STUDIES ON HEMOLYTIC SUBSTANCE PRODUCED BY Pseudomonas cepacia

洋葱假单胞菌产生溶血物质的分子和遗传学研究

• 批准号: 03670216
• 负责人: NAKAZAWA Teruko
• 金额: $ 1.34万
• 依托单位: YAMAGUCHI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670216/
• 关键词: Pseudomonas cepacia; small hemolytic substance; cepalycin; antifunfal activity; transposon mutagenesis; cepalycin-producing genes; DNA fingerprinting; nosocomial infection; cepalycin 産生遺伝子; 生理活性物質; 溶血活性; 遺伝子クロ-ニング

1)We established the method for purification of heat-stable hemolytic substance from P.cepacia JN106. Cell-washed supernatant was adsorbed to Amberlite XAD2,eluted with 70% ethanol containing 1mM HCI, and applied on reverse-phase HPLC. Two hemolytic substances,designated cepalycin I and cepalycin II, were isolated. 2)Cepalycins showed the same UV spectra,but the specific activity of cepalycin II was 4 times higher than that of cepalycin I. By LC-FAB Mass analysis,cepalycin II has the molecular weight of 240 with an OH group.Cepalycins were inactivated by alkaline,but resistant to acid.According to the solubility of various solvents,they appeared to be amphipathic molecules. 3)Cepalycins had antifungal activity comparable to amphotericin B. 4)Doughnut-like structures were observed by electron microscopy on a cepalycin-treated membrane of sheep erythrocytes,suggesting the pore-forming activity of the molecule. 5)Four types of non-hemolytic mutants of the strain JN106 were obtained by transposon mutagenesis. DNA fragments of JN106 were cloned that conferred the hemolytic activity to the mutants. 6)Distribution of cepalycin-producing genes were screened among the clinical and environmental isolates of P.cepacia by using the DNA fragment adjacent to the transposon of each mutant as a probe. One of the genes were found to be specific and exclusively for hemolytic strains. Thus ceplaycin is widely distributed among hemolytic strains of P.cepacia. 7)We have carried out DNA fingerprinting of P.cepacia to analyze a nosocomial outbreak of respiratory infection,and proved that the infection was associated with contaminated nebulizer devices.

1）建立了洋葱疫霉JN 106热稳定溶血物质的纯化方法。将细胞洗涤的上清液吸附到Amberlite XAD 2上，用含有ImM HCl的70%乙醇洗脱，并应用于反相HPLC。分离出两种溶血物质，命名为cepalycin I和cepalycin II。2)两种头孢菌素的紫外光谱基本一致，但头孢菌素II的比活性比头孢菌素I高4倍。LC-FAB质谱分析表明，Cepalycin II的分子量为240，分子中含有一个羟基，碱不活化，但耐酸，根据其在各种溶剂中的溶解度，推测其为两亲性分子。3)Cepalycins的抗真菌活性与异丙甲素B相当。（4）电镜观察到头孢菌素处理的绵羊红细胞膜上有环形结构，表明头孢菌素具有致孔活性。5)通过转座子诱变，获得了JN 106菌株的4种非溶血突变株。克隆JN 106的DNA片段，其赋予突变体溶血活性。6)以各突变体转座子附近的DNA片段为探针，筛选洋葱青霉临床分离株和环境分离株中产洋葱霉素基因的分布。其中一个基因被发现是特异性的，专门用于溶血菌株。因此，ceplaycin广泛分布在溶血性菌株的洋葱疫霉。7）对一起医院内呼吸道感染暴发疫情进行洋葱伯克霍尔德菌DNA指纹分析，证实感染与雾化器污染有关。

项目成果

Takigawa K,Fujita J,Negayama K,Yamagishi Y,Yamaji Y,Ouchi K,Yamada K,Abe M,Nakazawa T: "Nosocomial outbreak of Pseudomonas cepacia respiratory infection in immunocompromized patients with contaminated nebulizer devices" American Review of Respiratory Dise

Takikawa K、Fujita J、Negayama K、Yamagishi Y、Yamaji Y、Ouchi K、Yamada K、Abe M、Nakazawa T：“免疫功能低下患者因雾化器装置受污染而发生洋葱假单胞菌呼吸道感染的医院爆发”美国呼吸疾病评论

Takigawa K, Fujita J, Negayama K,Yamagishi Y, Yamaji Y,Ouchi K,Yamada K,Abe M, Nakazawa T: "Nosocomial outbreak of Pseudomonas cepacia respiratory infection in immunocompromized patients with contaminated nebulizer devices" American Review of Respiratory

Takikawa K、Fujita J、Negayama K、Yamagishi Y、Yamaji Y、Ouchi K、Yamada K、Abe M、Nakazawa T：“免疫功能低下患者因雾化器装置受污染而发生洋葱假单胞菌呼吸道感染的院内爆发”美国呼吸系统评论

阿部 光子、津田 雅孝、中澤 晶子: "P.cepaciaの産生する溶血物質の精製とその生合成遺伝子" 日本細菌学会誌. 46. 183- (1991)

Mitsuko Abe、Masataka Tsuda、Akiko Nakazawa：“洋葱假单胞菌及其生物合成基因产生的溶血产物的纯化”日本细菌学会杂志 46. 183- (1991)。

西口 まゆみ、川瀧 美智子、久保田 小百合、瀧川 圭一、山岸 善文、根ヶ山 清、藤田 次郎、森田 敏子、中澤 晶子: "P.cepacia院内感染症の感染源の追及と対策" 環境感染. 7. 33-37 (1992)

Mayumi Nishiguchi、Michiko Kawataki、Sayuri Kubota、Keiichi Takikawa、Yoshifumi Yamagishi、Kiyoshi Negayama、Jiro Fujita、Toshiko Morita、Akiko Nakazawa：“追寻感染源和医院内 P.cepacia 感染的对策” 环境感染。 37 (1992)

Abe M, Nakazawa T: "Isolation and characterization of new hemolytic substance,cepalycin, from Pseudomonas cepacia"

Abe M、Nakazawa T：“洋葱假单胞菌中新溶血物质头孢霉素的分离和表征”