Studies in membrane topology and function of inner membrane component, MexA,of antibiotic extrusion pump of Pseudomonas aeruginosa

铜绿假单胞菌抗生素挤出泵膜拓扑结构及内膜成分MexA功能研究

• 批准号: 09670298
• 负责人: YONEYAMA Hiroshi
• 金额: $ 1.66万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670298/
• 关键词: Pseudomonas aeruginosa; multidrug resistance; extrusion pump; inner membrane protein; outer membrane protein; antibiotics; 他剤耐性

Pseudomonas aeruginosa, a main causative agent of opportunistic infections, shows intrinsic and multiantibiotic resistance. The major reason for the antibiotic resistance in this bacterium appeared to be the presence of an energy dependent antibiotic extrusion pump (MexAB-OprM pump) encoded in the mex operon (mexA-mexl3-oprM). To get insight into the mode of antibiotic extrusion by the Mex pump, it is essential to study on the structure and function of each subunit of the pump. Therefore, I analyzed the membrane topology and localization of MexA, a member of membrane fusion protein family, which is thought to link inner membrane pump, MexB, to an outer membrane channel, OprM.I employed the gene fusion technique to analyze the membrane topology of MexA using signal sequenceless beta-lactamase as a reporter and got 78 in-frame MexA-beta--lactamase fusions. All transformants harbored each fusion gene showed ampicillin resistance, indicating that almost all regions of MexA beyond the signal sequence reside in the periplasm. MexA was also found to be modified by lipid(s) at its cysteine residue in the consensus sequence for lipoproteins.

铜绿假单胞菌是机会性感染的主要病原体，表现出内在的多抗生素耐药性。该细菌产生抗生素耐药性的主要原因似乎是 mex 操纵子 (mexA-mexl3-oprM) 中编码的能量依赖性抗生素挤出泵 (MexAB-OprM 泵) 的存在。为了深入了解Mex泵的抗生素挤出模式，有必要研究泵各亚基的结构和功能。因此，我分析了膜融合蛋白家族成员 MexA 的膜拓扑和定位，MexA 被认为将内膜泵 MexB 与外膜通道 OprM 连接起来。我采用基因融合技术来分析膜拓扑使用无信号序列 β-内酰胺酶作为报告基因对 MexA 进行编码，得到 78 个框内 MexA-β-内酰胺酶融合体。所有携带每个融合基因的转化体均表现出氨苄青霉素抗性，表明信号序列之外的几乎所有 MexA 区域都位于周质中。还发现 MexA 在脂蛋白共有序列中的半胱氨酸残基处被脂质修饰。

项目成果

Kitahara, T., Yoneyama, H.and Nakae, T.: "Antibiotic diffusion pathways in the outer membrane of Pseudomonas aeruginosa." Biochem.Biophys.Res.Commun.238. 457-461 (1997)

Kitahara, T.、Yoneyama, H. 和 Nakae, T.：“铜绿假单胞菌外膜中的抗生素扩散途径。”

Kitahara et al.: "Antibiotic diffusion pathways in the outer membrane of Pseudomonas aeruginosa." Biochem.Biophys.Res.Commun.238. 457-461 (1997)

Kitahara 等人：“铜绿假单胞菌外膜中的抗生素扩散途径。”

Ocaktan et al.: "Use of fluorescence probes to monitor function of the subunit proteins of the MexA- MexB- OporM drug extrusion machinery in Pseudomonas aeruginosa." J.Biol.Chem.272. 21964-21969 (1997)

Ocaktan 等人：“使用荧光探针监测铜绿假单胞菌中 MexA-MexB-OporM 药物挤出机械亚基蛋白的功能。”

Ocaktan, A., Yoneyama, H.and Nakae, T.: "Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-OporM drug extrusion machinery in Pseudomonas aeruginosa." J.Biol.Chem.272. 21964-21969 (1997)

Ocaktan, A.、Yoneyama, H. 和 Nakae, T.：“使用荧光探针监测铜绿假单胞菌中 MexA-MexB-OporM 药物挤出机械亚基蛋白的功能。”

Yoneyama et al.: "Subunit swapping in the Mex-extrusion pumps in Pseudomonas aeruginosa." Biochem.Biophys.Res.Commun.244. 898-902 (1998)

Yoneyama 等人：“铜绿假单胞菌 Mex 挤出泵中的亚基交换。”