Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes.

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.

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

A Unified Framework for the Incorporation of Bioorthogonal Compound Exposure Probes within Biological Compartments.

Compartmentalization is a crucial facet of many biological systems, and key aspects of cellular processes rely on spatial segregation within the cell. While many drug targets reside in specific intracellular compartments, the tools available for assessing compound exposure are generally limited to whole-cell measurements. To address this gap, we recently developed a bioorthogonal chemistry-based method to assess compartment-specific compound exposure and demonstrated its use in Gram-negative bacteria. To expand the applicability of this approach, we report here novel bioorthogonal probe modalities which enable diverse probe incorporation strategies. The probes we developed utilize a cleavable thiocarbamate linker to connect localizing elements such as metabolic substrates to a cyclooctyne moiety which enables the detection of azide-containing molecules. Adducts between the probe and azide-bearing compounds can be recovered and affinity purified after exposure experiments, thus facilitating the mass-spectrometry based analysis used to assess compound exposure. The bioorthogonal system reported here thus provides a valuable new tool for interrogating compartment-specific compound exposure in a variety of biological contexts while retaining a simple and unified sample preparation and analysis workflow.

Targeted lipopolysaccharide biosynthetic intermediate analysis with normal-phase liquid chromatography mass spectrometry.

Lipopolysacharride (LPS) forms the outer leaflet of the outer membrane in Gram-negative bacteria and contributes to the permeability barrier and immune response. In this study, we established a method for monitoring the LPS biosynthetic intermediates of the Raetz pathway (lpxA-lpxK) in Escherichia coli. Metabolites from compound-treated cells and genetically-perturbed cells were extracted from whole cells and concentrated by mixed-mode weak anion exchange (WAX) solid-phase extraction (SPE) prior to analysis by normal phase (NP)LC-MS/MS. Data was normalized to cell density and an internal standard prior to comparison against untreated cells in order to determine fold accumulation and depletion for affected metabolites. Using this LC-MS/MS method, we were able to reliably monitor changes in levels of the LPS intermediates in response to compound-treatment and genetic modification. In addition, we found that deletion of periplasmic CDP-diacylglycerol pyrophosphatase dramatically increased levels of the UDP-containing LPS intermediates, suggesting the enzymatic breakdown during sample preparation. This assay allows for probing a key essential pathway in Gram-negative bacteria in an effort to discover antibacterial agents that inhibit enzymes in the LPS biosynthetic pathway.