Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli
Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli
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DOI：
10.1128/jb.187.6.1923-1929.2005
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发表时间：
2005-03-01
影响因子：
3.2
通讯作者：
Nikaido, H
中科院分区：
文献类型：
--
作者：
Aires, JR;Nikaido, H
为了更好地了解耐药结节分裂（RND）型多药外排泵的机制，我们检查了大肠杆菌 AcrD 泵，其典型底物氨基糖苷类预计不会自发扩散穿过脂质双层。六组氨酸标记的 AcrD 蛋白被纯化并重构为单层蛋白脂质体。其活性通过伴随底物传输的质子通量来测量。当蛋白脂质体内部酸化时，向外部介质中添加氨基糖苷类会刺激质子流出和放射性标记庆大霉素的囊泡内积累，这表明氨基糖苷类可以从该系统的外部介质（对应于细胞质）中被捕获和转运。该活性需要蛋白脂质体内存在 AcrA。有趣的是，当氨基糖苷类仅存在于囊泡内时，质子流出也会增加。这一结果表明，AcrD 还可以从周质捕获氨基糖苷类药物，将其挤出到完整细胞的培养基中，充当“周质真空吸尘器”。
To understand better the mechanisms of resistance-nodulation-division (RND)-type multidrug efflux pumps, we examined the Escherichia coli AcrD pump, whose typical substrates, aminoglycosides, are not expected to diffuse spontaneously across the lipid bilayer. The hexahistidine-tagged AcrD protein was purified and reconstituted into unilamellar proteoliposomes. Its activity was measured by the proton flux accompanying substrate transport. When the interior of the proteolliposomes was acidified, the addition of aminoglycosides to the external medium stimulated proton efflux and the intravesicular accumulation of radiolabeled gentamicin, suggesting that aminoglycosides can be captured and transported from the external medium in this system (corresponding to cytosol). This activity required the presence of AcrA within the proteolliposomes. Interestingly, the increase in proton efflux illso occurred when aminoglycosides were present only in the intravesicular space. This result suggested that AcrD can also capture aminoglycosides from the periplasm to extrude them into the medium in intact cells, acting as a "periplasmic vacuum cleaner".