1. Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics.
- Author
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Werneburg M, Zerbe K, Juhas M, Bigler L, Stalder U, Kaech A, Ziegler U, Obrecht D, Eberl L, and Robinson JA
- Subjects
- Anti-Bacterial Agents chemistry, Bacterial Outer Membrane Proteins antagonists & inhibitors, Bacterial Outer Membrane Proteins genetics, Biological Transport drug effects, Biological Transport genetics, Carbon Radioisotopes, Cell Membrane metabolism, Escherichia coli, Genetic Complementation Test, Lipid A chemistry, Lipid A metabolism, Lipopolysaccharides antagonists & inhibitors, Microscopy, Electron, Molecular Structure, Mutation, Peptidomimetics chemistry, Periplasm metabolism, Promoter Regions, Genetic, Pseudomonas aeruginosa genetics, Pseudomonas aeruginosa metabolism, Transfection, Anti-Bacterial Agents pharmacology, Bacterial Outer Membrane Proteins metabolism, Cell Membrane drug effects, Lipopolysaccharides metabolism, Peptidomimetics pharmacology, Periplasm drug effects, Pseudomonas aeruginosa drug effects
- Abstract
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., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2012
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