Chemistry Department, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Chembiochem. 2012 Aug 13;13(12):1767-75. doi: 10.1002/cbic.201200276. Epub 2012 Jul 17.
The asymmetric outer membrane (OM) of Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet and phospholipid in the inner leaflet. During OM biogenesis, LPS is transported from the periplasm into the outer leaflet by a complex comprising the OM proteins LptD and LptE. Recently, a new family of macrocyclic peptidomimetic antibiotics that interact with LptD of the opportunistic human pathogen Pseudomonas aeruginosa was discovered. Here we provide evidence that the peptidomimetics inhibit the LPS transport function of LptD. One approach to monitor LPS transport involved studies of lipid A modifications. Some modifications occur only in the inner membrane while others occur only in the OM, and thus provide markers for LPS transport within the bacterial envelope. We prepared a conditional lptD mutant of P. aeruginosa PAO1 that allowed control of lptD expression from the rhamnose promoter. With this mutant, the effects caused by the antibiotic on the wild-type strain were compared with those caused by depleting LptD in the mutant strain. When LptD was depleted in the mutant, electron microscopy revealed accumulation of membrane-like material within cells and OM blebbing; this mirrored similar effects in the wild-type strain caused by the antibiotic. Moreover, the bacterium responded to the antibiotic, and to depletion of LptD, by introducing the same lipid A modifications, consistent with inhibition by the antibiotic of LptD-mediated LPS transport. This conclusion was further supported by monitoring the radiolabelling of LPS from [¹⁴C]acetate, and by fractionation of IM and OM components. Overall, the results provide support for a mechanism of action for the peptidomimetic antibiotics that involves inhibition of LPS transport to the cell surface.
革兰氏阴性菌的不对称外膜（OM）在外层含有脂多糖（LPS），在内层含有磷脂。在 OM 生物发生过程中，LPS 通过由 OM 蛋白 LptD 和 LptE 组成的复合物从周质转运到外层。最近，发现了一类与机会性病原体铜绿假单胞菌的 LptD 相互作用的新型大环肽拟肽抗生素。本文提供了证据表明这些肽拟肽抑制了 LptD 的 LPS 转运功能。一种监测 LPS 转运的方法涉及脂质 A 修饰的研究。一些修饰仅发生在内膜中，而另一些修饰仅发生在外膜中，因此为 LPS 在细菌包膜内的转运提供了标记。我们制备了铜绿假单胞菌 PAO1 的条件性 lptD 突变体，允许从鼠李糖启动子控制 lptD 的表达。利用该突变体，比较了抗生素对野生型菌株的影响与抗生素在突变菌株中耗尽 LptD 时的影响。当 LptD 在突变体中耗尽时，电子显微镜显示细胞内和 OM 泡状结构中积累了膜状物质；这与抗生素在野生型菌株中引起的类似效应相吻合。此外，细菌通过引入相同的脂质 A 修饰对抗生素和 LptD 的耗竭做出反应，这与抗生素抑制 LptD 介导的 LPS 转运一致。通过监测来自 [¹⁴C]乙酸盐的 LPS 的放射性标记以及对 IM 和 OM 成分的分级分离，进一步支持了这一结论。总的来说，这些结果为肽拟肽抗生素的作用机制提供了支持，该机制涉及抑制 LPS 向细胞表面的转运。
