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
| Project/Area Number |
17590402
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Tokai University |
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Principal Investigator |
YOSHIHARA Eisaku Tokai University, School of Medicine, Associate Professor, 医学部, 助教授 (70167063)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2005: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Pseudomonas aeruginosa / multidrug efflux pump / MexAB-OprM / outer membrane channel / oligomeric structure / extracellular loops / inhibitors / monoclonal antibodies / 外服チャネル / 阻害物質 / 院内感染 / 多剤耐性 / 薬剤排出ポンプ / チャネル / 薬剤感受性 |
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| Research Abstract |
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
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Report
(3 results)
Research Products
(11 results)
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[Journal Article] Multidrug transporter MexB of Pseudomonas aeruginosa : overexpression, purification, and initial structural characterization2005
Author(s)
Mokohonov, V., Mokohonova, E, Yoshihara, E., Masui, R., Sakai, M., Akama, H., Nakae, T.
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Journal Title
Protein Expr.Purif. 40
Pages: 91-100
Description
「研究成果報告書概要(欧文)」より
Related Report
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