Inhibition of lipopolysaccharide transport to the outer membrane in Pseudomonas aeruginosa by peptidomimetic antibiotics.
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.
Chemistry Department, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Martina
M
Werneburg
Katja
K
Zerbe
Mario
M
Juhas
Laurent
L
Bigler
Urs
U
Stalder
Andres
A
Kaech
Urs
U
Ziegler
Daniel
D
Obrecht
Leo
L
Eberl
John A
JA
Robinson
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001425:Bacterial Outer Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D004926:Escherichia coli; D005816:Genetic Complementation Test; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008854:Microscopy, Electron; D015394:Molecular Structure; D009154:Mutation; D057786:Peptidomimetics / Q000737:chemistry / Q000494:pharmacology*; D019897:Periplasm / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D002250:Carbon Radioisotopes; D008050:Lipid A; D008070:Lipopolysaccharides; D057786:Peptidomimetics