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Discovery of the extracellular loops being essential for the proper function of the multidrug efflux pump suggests that the loops may be an excellent target for the pump inhibitor

细胞外环的发现对于多药物外排泵的正常功能至关重要，这表明环可能是泵抑制剂的极好靶标

基本信息

批准号：
17590402
负责人：
YOSHIHARA Eisaku
金额：
$ 2.24万
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：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Pseudomonas aeruginosa, an opportunistic pathogen, exhibits a high-level resistance to a wide variety of antibiotics that is mainly mediated by the multidrug efflux pumps in this organism. Although several RND efflux pump genes are coded in the P. aeruginosa genome, MexAB-OprM gene is only constitutively expressed. MexAB-OprM is a multicomponent efflux pump : MexB functions as a drug/proton antiporter, OprM as a channel for drugs in the outer membrane, and MexA as a linker between MexB and OprM. We focused on the OprM channel and examined its function and structure.(1)We examined the oligomeric structure of OprM and its homologs such as OprJ and OprN by crosslinking. OprM and OprN were shown to form a trimer, but unexpectedly OprJ was found to form a tetramer, indicating that homologous outer membrane channels with the same function can assume different oligomeric structures.(2)OprM (468 amino acids) consists of the three domains (α-helical periplasmic domain, β-barrel transmembrane do … More main and two extracellular loop (EL)), and two ELs consist of P100〜P109 (EL1) and R311〜G320 (EL2), respectively. In order to elucidate the function of the EL1 and EL2, we exchanged the amino acid residues of these ELs to Cys and examined the activity of MexAB-mutant OprM. It was clearly demonstrated that the mutation in the EL1 or EL2 caused the dysfunction of the pump, indicating that both EL1 and EL2 play the pivotal role in the drug extrusion by the pump. Furthermore, to be surprised, EL1 and EL2 are shown to be the sites contributing to the substrate recognition by the pump.(3)Based on the above results, we considered that EL might become an excellent target site for the inhibitors of the efflux pump since the inhibition of the EL function could lead to the dysfunction of the pump. As EL is accessible from the outside of the cell, we though that the monoclonal antibody (mAb) can be used to interfere the EL function. Then we synthesized the peptide having the same amino acid sequence as that of EL2 and used it as an antigen. We generated the hybridomas and then their supernatants were subjected to ELISA to test the binding activity to the peptide. Up to now, we could produce 17 hybridomas secreting mAb with the binding activity to the peptide. Less

铜绿假单胞菌是一种条件致病菌，对多种抗生素具有高水平的耐药性，其耐药性主要由该菌体内的多药外排泵介导。虽然铜绿假单胞菌基因组中编码几个RND外排泵基因，但MexAB-OprM基因仅组成型表达。MexAB-OprM是一种多组分外排泵：MexB作为药物/质子反向转运体，OprM作为外膜中药物的通道，MexA作为MexB和OprM之间的连接体。我们关注OprM渠道，并研究其功能和结构。(1)We通过交联研究了OprM及其同系物如OprJ和OprN的低聚结构。OprM和OprN显示形成三聚体，但意外地发现OprJ形成四聚体，表明具有相同功能的同源外膜通道可以呈现不同的寡聚体结构。（2）OprM由三个结构域（α-螺旋周质结构域、β-桶形跨膜结构域）组成，共468个氨基酸。 ...更多信息主要的和两个胞外环（EL）），和两个EL分别由P100 → P109（EL 1）和R311 → G320（EL 2）组成。为了阐明EL 1和EL 2的功能，我们将这些EL的氨基酸残基交换为Cys，并检测MexAB突变体OprM的活性。结果表明，EL 1或EL 2基因突变可导致泵的功能障碍，说明EL 1和EL 2基因在泵的药物挤出中起着关键作用。此外，令人惊讶的是，EL 1和EL 2显示为有助于通过泵识别底物的位点。（3）根据上述结果，我们认为EL可能成为外排泵抑制剂的一个很好的靶点，因为EL功能的抑制可能导致外排泵的功能障碍。由于EL可以从细胞外进入，我们认为单克隆抗体（mAb）可以用来干扰EL功能。然后，我们合成了具有与EL 2相同的氨基酸序列的肽，并将其用作抗原。我们产生了杂交瘤，然后将其上清液进行ELISA以测试与肽的结合活性。到目前为止，我们已经获得了17株分泌具有结合活性的单克隆抗体的杂交瘤。少