The xenobiotic efflux pumps and multiantibiotic resistance in Pseudomonas aeruginosa

铜绿假单胞菌的异生素外排泵和多抗生素耐药性

• 批准号: 09470079
• 负责人: NAKAE Taiji
• 金额: $ 5.38万
• 依托单位: Tokai University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09470079/
• 关键词: Antibiotic resistance; Pump; Membrane protein; Membrane topology; Membrane; Transport; Pseudomonas; Efflux; 外膜; 異物; 抗生物質; 耐性; 異物排出; 生体防御

Pseudomonas aeruginosa is a major pathogen in the hospital that exhibits resistance to many structurally and functionally diverse antibiotics, This multidrug resistance is mainly attributable to a tight outer membrane permeability and expression of the xenobiotic efflux pumps. The MexAB-OprM efflux pump consists of three subunits such as inner membrane protein, MexB, inner membrane associated periplasmic protein, MexA and outer membrane protein, OprM, functioning as the energy dependent efflux pump, the membrane fusion protein and the xenobiotic exit channel, respectively, We studied structure and function of these pump subunit proteins. (I) To acess the role of the subunit proteins in the antibiotic export, we constructed by mean of gene replacement technique the mutant lacking either MexA, MexB, OprM or all combinations of these subunits. The antibiotic susceptibility test of these mutant revealed that the mutants lacking MexA or MexB are equally hypersusceptible to many antibiotics, … More whereas the mutants lacking the OprM subunit are more antibiotic hypersusceptible than the MexA or MixB mutants. (ii) To quantitate the pump function in real time, we developed the method to determine the fluorescent dye accumulation in stead of measuring the antibiotic susceptibility. The dyes accumulation in the cell was proportionally related with the antibiotic susceptibility of the cells. Thus, the measurement of fluorescent dye accumulation could be used as a pump function monitor. (iii) P. aeruginosa encodes MexCD-OprJ pump upon the regulator mutation, the nfxB gene. Since the amino acid sequence of the MexAB-OprM and MexCD-OprJ has high similarity, we tested a possible interchangeability of the pump subunits, expressing the hybrid pump. To do it, OprJ subunit, for instance, was expressed in the host lacking OprM subunit and producing the MexA and MexB subunits. Results of such study revealed that the outer membrane subunit could be substituted, but not the inner membrane subunit. Thus, it became clear that P. aeruginosa expressing more than two pump systems produces the hybrid pumps. (iv) The MexB subunit is assumed to be the energy dependent efflux pump crossing the cytoplasmic membrane several times. However, there is no experimental manifestation to this assumption. We studied the membrane topology of the MexB protein by the reporter gene (phoA ) fusion method to the various length of carboxyl terminal truncated mexB gene. Expression of such fusion genes showed that the alkaline phosphatase activity of the fusion protein, of which fusion-site is faced to the periplasm would be high. On one hand, the enzyme activity would be undetectably low in the hybrid protein, of which reporter gene is located in the cytoplasm. We constructed a total 28 hybrid proteins. The result showed that the MexB protein bears 12 transmembrane domains leaving the amino and carboxyl termini in the cytoplasm. Interestingly, the MexB protein had two large hydrophilic domains containing 311 and 314 amino acid residues. Thus, it was suggested that these two large domains may interact with the MexA and Oprm subunits. Less

铜绿假单胞菌是医院感染的主要病原菌，对多种结构和功能多样的抗生素具有耐药性，这种多药耐药主要归因于紧密的外膜通透性和外源性外排泵的表达。MexAB-OprM外排泵由内膜蛋白MexB、内膜相关周质蛋白MexA和外膜蛋白OprM三个亚基组成，分别作为能量依赖性外排泵、膜融合蛋白和外源物质出口通道。(I)为了探讨亚基蛋白在抗生素输出中的作用，我们利用基因置换技术构建了缺失MexA、MexB、OprM或所有这些亚基组合的突变体。对这些突变株的药敏试验表明，缺失MexA或MexB的突变株对多种抗生素同样敏感， ...更多信息 而缺少OprM亚基的突变体比MexA或MixB突变体更易对抗生素过敏。(ii)为了在真实的时间内定量泵功能，我们开发了测定荧光染料积累的方法来代替测量抗生素敏感性。染料在细胞中的积累与细胞对抗生素的敏感性成正比。因此，荧光染料积累的测量可以用作泵功能监测器。(iii)铜绿假单胞菌在调节子突变nfxB基因后编码MexCD-OprJ泵。由于MexAB-OprM和MexCD-OprJ的氨基酸序列具有高度相似性，我们测试了表达杂合泵的泵亚基的可能的可重复性。为此，例如，OprJ亚基在缺乏OprM亚基并产生MexA和MexB亚基的宿主中表达。研究结果表明，外膜亚基可以被取代，但内膜亚基不能被取代。因此，很明显，表达两个以上泵系统的铜绿假单胞菌产生混合泵。(iv)MexB亚基被认为是穿过细胞质膜几次的能量依赖性外排泵。然而，没有实验证明这一假设。我们研究了膜拓扑结构的MexB蛋白的报告基因（phoA）融合的方法，以不同长度的羧基端截短的mexB基因。这些融合基因的表达表明，融合位点朝向周质的融合蛋白碱性磷酸酶活性高。一方面，在杂合蛋白中，酶活性将是不可检测的低，其报告基因位于细胞质中。我们共构建了28个杂交蛋白。结果表明，MexB蛋白具有12个跨膜结构域，氨基和羧基末端留在胞质中。有趣的是，MexB蛋白有两个大的亲水性结构域，分别含有311和314个氨基酸残基。因此，这表明这两个大结构域可能与MexA和Oprm亚基相互作用。少

项目成果

Monika Germ, Eisaku Yoshihara, Hiroshi Yoneyama and Taiji Nakae: "Interplay between the efflux pump and the outer membrane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 261. 452-455 (1999)

Monika Germ、Eisaku Yoshihara、Hiroshi Yoneyama 和 Taiji Nakae：“铜绿假单胞菌荧光染料积累中外排泵和外膜渗透性屏障之间的相互作用”Biochem。

Taiji Nakae: "Interplay between the efflux pump and the outer memberane permeability barrier in fluorescent dye accumulation in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 261. 452-455 (1999)

Taiji Nakae：“铜绿假单胞菌荧光染料积累中外排泵和外膜渗透性屏障之间的相互作用”Biochem。

Taiji Nakae: "Identification of the Catalytic Triad of the Protein D2 Protease in Pseudomonas aeruginosa" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 247. 142-145 (1998)

Taiji Nakae：“铜绿假单胞菌中蛋白质 D2 蛋白酶催化三联体的鉴定”生物化学和生物物理研究通讯。

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

Nakae,T.：“铜绿假单胞菌外膜中的抗生素扩散途径”Biochem.Biophys.Res.Commun.238。

Keiko Suzuki: "Subunit Swapping in the Mex-Extrusion Pumps in Pseudomonas aeruginosa"Biochem. Biophys. Res. Commun.. 244. 898-902 (1998)

Keiko Suzuki：“铜绿假单胞菌 Mex 挤出泵中的亚基交换”Biochem。