Nilsson, Inga, Lee, Sheng Y., Sawyer, William S., Baxter Rath, Christopher M., Lapointe, Guillaume and Six, David A. (2019) Metabolic labeling of phospholipids in intact bacteria enables a fluorescence assay to detect loss of outer membrane asymmetry. ACS infectious diseases.

Abstract

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPS) on the outer leaflet and phospholipids (PLs) on the inner leaflet. Loss of asymmetry due to mutation in the lipopolysaccharide (LPS) biosynthesis or transport pathways causes PLs to be externalized to the outer leaflet of the OM and leads to OM permeability defects. Here, we employ metabolic labeling to detect externalized PLs on intact bacteria. Phosphatidylcholine synthase (Pcs) expression in Escherichia coli allowed for incorporation of exogenous propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC) and the incorporation of exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC) as confirmed by liquid chromatography-mass spectrometry (LC-MS). AEPC readily reacted with a fluorescent copper-free click reagent in lysed cells, but poorly labeled intact wild-type cells. Fluorescence microscopy and flow cytometry analysis confirmed significantly higher levels of externalized AEPC were present on an E. coli LPS transport mutant (lptD4213) and a LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool to detect PL externalization, dissect the mechanisms of PL retrograde and anterograde transport, and identify or characterize novel cell-active inhibitors of LPS biosynthesis or transport.

Item Type:	Article
Keywords:	Phospholipids, Phospholipids/Phosphatidylcholine, Phospholipids/biosynthesis, Phospholipid/trafficking, membranes, metabolic labeling, choline, lptD, lpxC, phospholipid externalization
Date Deposited:	10 Dec 2019 00:45
Last Modified:	10 Dec 2019 00:45
URI:	https://oak.novartis.com/id/eprint/39687