Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes[S]

摘要

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.