Analysis of MexA/MexB substrate recognition complex of antibiotic efflux pump of Pseudomonas aeruginosa

铜绿假单胞菌抗生素外排泵MexA/MexB底物识别复合物分析

• 批准号: 11670274
• 负责人: YONEYAMA Hiroshi
• 金额: $ 2.24万
• 依托单位: Tohoku University (2000)Tokai University (1999)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670274/
• 关键词: Pseudomonas aeruginosa; antibiotic resistance; memebrane protein; efflux pump; tranporter; opportunistic infection; random mutagenesis; membrane fusion protein family; 多剤耐性; 抗生物質; 内臓タンパク; ペリプラズムタンパク

A major cause of antibiotic resistance of Pseudomonas aeruginosa has recently appeared to be the energy-dependent antibiotic efflux pump, MexA,B-OprM.Analysis of chimeric pump between MexA,B-OprM and its homologous counterpart, MexC,D-OprJ, suggests that MexA and MexB form a substrate recognition complex. In this study, membrane topology of inner membrane subunits, MexA and MexB, were analyzed by gene-fusion technique, and MexB mutants were obtained by random mutagenesis method. These experiments shows : (1) MexA has lipid modification at the 24th cystein residue and anchors inner membrane through the lipid. The region beyond the cystein residue localizes in the periplasm. (2) The hydrophilic periplasmic region of MexA is important, but lipid modification is not essential for function of the MexA.(3) MexB laas 12 trans membrane segments (TMS) and the large 1st and 4th loops with about 300 amino acid residues located in the periplasm. (4) MexB mutants obtained by random mutagenesis has mutations in the cytoplasmic loop, TMS, and periplasmic loops. (5) Suppressor mutants obtained from the MexB mutant having a mutation in the TMS10 have the same second mutation in TMS4. From these results, TMS4 and TMS10 are suggested to pack in the close vicinity in inner membrane and the periplasmic loops 1 and 4 might interact with MexA to form substrate recognition complex.

铜绿假单胞菌抗生素抗性的主要原因最近似乎是能量依赖性的抗生素外排泵，MEXA，B-oprm.Mexa，B-oprm及其同源物，Mexc，Mexc，d-oprj之间的嵌合泵的分析MEXA和MEXB形成了底物识别综合体。在这项研究中，通过基因融合技术分析了内膜亚基，MEXA和MEXB的膜拓扑，并通过随机诱变方法获得了MEXB突变体。这些实验表明：（1）MEXA在第24 Cystein残留物处具有脂质修饰，并通过脂质锚固内膜。 Cystein残留物以外的区域位于周质中。 （2）MEXA的亲水性周质区域很重要，但是脂质修饰对于MEXA的功能并不是必需的。（3）MEXB LAAS 12反式膜段（TMS）和大约300氨基酸残基在周期中。 （4）通过随机诱变获得的MEXB突变体在细胞质环，TM和周质环中具有突变。 （5）从TMS10中具有突变的MEXB突变体获得的抑制突变体在TMS4中具有相同的第二突变。从这些结果中，建议TMS4和TMS10在内膜附近包装，而周环路1和4可能与MEXA相互作用以形成底物识别复合物。

项目成果

Yoneyama et al.: "Function of the membrane fusion protein, MexA, of the MexA, B-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane"J.Biol.Chem.. 275. 4628-4634 (2000)

Yoneyama 等：“无锚定膜的铜绿假单胞菌中 MexA、B-OprM 外排泵的膜融合蛋白 MexA 的功能”J.Biol.Chem.. 275. 4628-4634 (2000)

Maese et al.: "Assignment of the substrate-selective subunits of the MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa"Antimicrob.Agents Chemother.. 44. 658-664 (2000)

Maese 等人：“铜绿假单胞菌 MexEF-OprN 多药外排泵的底物选择性亚基的分配”Antimicrob.Agents Chemother.. 44. 658-664 (2000)

Yoneyama et al.: "Function of the membrane fusion protein, MexA, of the MexA, B-OprM efflux pump in Pseudomonas aeruginosa without an anchoring membrane"J.Biol Chem.. 275. 4628-4634 (2000)

Yoneyama 等：“无锚定膜的铜绿假单胞菌中 MexA、B-OprM 外排泵的膜融合蛋白 MexA 的功能”J.Biol Chem.. 275. 4628-4634 (2000)

Germ, et al.: "Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accululation in Pseumonas aeruginosa"Biochem. Biophys. Res. Commun.. 261. 452-455 (1999)

Germ 等人：“铜绿假单胞菌荧光染料积聚中外排泵与外膜渗透性屏障之间的相互作用”Biochem。

Guan et al.: "Membrane topology of the xenobiotic-exporting subuint, MexB, of the MexA, B-OprM extrusion pump in Pseudomonas aeruginosa"J.Biol.Chem.. 274. 10517-10522 (1999)

guan 等人：“铜绿假单胞菌中 MexA、B-OprM 挤出泵的异生素输出亚单位 MexB 的膜拓扑”J.Biol.Chem.. 274. 10517-10522 (1999)