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The permeability specificity of the imipenem-specific channel of OprD2 in Pseudomonas aeruginosa

铜绿假单胞菌中OprD2亚胺培南特异性通道的通透性特异性

基本信息

批准号：
05454195
负责人：
NAKAE Taiji
金额：
$ 2.5万
依托单位：
Tokai University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454195/
关键词：
Reconstitution Outer membrane Pseudomonas aeruginosa Lipid bilayr Porin Antibiotic resistance Channel Membrane protein ポ-リン Pseudomonas OprD2 イミペネム脂質平面膜

项目摘要

The outer membrane of Pseudomonas aeruginosa is barrier to the penetration of antibiotics and hence the bacteria is naturally resistant to many antibiotics. Imipenem and closely related carbapenem antibiotics are powerful to the therapy of Pseudomonas aeruginosa infection. The imipenem resistant mutants isolated was found to be lacking the outer membrane protein D2. Hence, it was assumed that protein D2 is responsible in forming the imipenem-permeable channel. Aim of this study is to characterize the specific interaction of imipenem (carbapenem) and the protein D2 porin. To investigate this, a strategy we took was as follows. Planar lipid bilayrs were formed in 0.1 M NaCl solution and to it was added a small amount of highly purified protein D2. We detected the conductivity increment and frequent flickering of about 100 pS in 0.1 M NaCl those are the open/close signal of protein D2 channel. We added 0.7 mM of imipenem to the bath solution and observed that the conductivity was lowered and the channel opening became less frequent. Imipenem at 4 mM completely closed the channel. We tested the effect of another carbapenem, DX8739, and found that this new carbapenem closed the protein D2 channel at about 1 mM.We interpreted these results that imipenem and DX8739 specifically bound to protein D2 and exerted the effect to close the channel. This is the first observation to our best knowledge that the antibiotic and porin channel interaction was determined as single molecular events.

铜绿假单胞菌的外膜是抗生素渗透的屏障，因此细菌对许多抗生素具有天然抗性。亚胺培南及与之密切相关的碳青霉烯类抗生素是治疗铜绿假单胞菌感染的有效药物。发现分离的亚胺培南抗性突变体缺乏外膜蛋白D2。因此，假定蛋白D2负责形成亚胺培南渗透通道。本研究的目的是表征亚胺培南（碳青霉烯）和蛋白质D2孔蛋白的特异性相互作用。为了研究这个问题，我们采取了如下策略。在0.1 M NaCl溶液中形成平面脂质双层，并向其中加入少量高度纯化的蛋白D2。在0.1 M NaCl溶液中，我们检测到了约100 pS的电导率增量和频繁的闪烁，这是蛋白D2通道的开/关信号。我们将0.7 mM亚胺培南加入到浴溶液中，观察到电导率降低，通道开放变得不那么频繁。4 mM的亚胺培南完全关闭通道。我们测试了另一种碳青霉烯类药物DX 8739的作用，发现这种新的碳青霉烯类药物在约1 mM时关闭蛋白D2通道。我们解释了这些结果，亚胺培南和DX 8739特异性地结合蛋白D2并发挥关闭通道的作用。据我们所知，这是第一次观察到抗生素和孔蛋白通道相互作用被确定为单分子事件。