A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in Acinetobacter baumannii

Abstract

Acinetobacter baumannii ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes lpxA, lpxC or lpxD. Although LOS itself is not essential for growth of A. baumannii ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.

Pushing the envelope: LPS modifications and their consequences

“…Inhibitors of the latter steps in lipid A/LPS synthesis could still prove useful in combination with drugs that normally cannot permeate the asymmetric OM. The most recent studies targeting LPS synthesis have attempted to increase the efficacy of LpxC inhibitors 107 , identify compounds that target LpxA through virtual screening methods 108 ,and develop new high-throughput screening strategies 109,110 for other Lpx enzymes (e.g. LpxH).…”

Deciphering the Metabolic Pathway Difference Between Saccharopolyspora pogona and Saccharopolyspora spinosa by Comparative Proteomics and Metabonomics

“…Fatty acid degradation metabolism and TCA cycle are important metabolic pathways for the synthesis of acetyl-CoA and other intermediate precursors, respectively (Edmunds et al, 2014;Vuoristo et al, 2017;Richie et al, 2018). Bacteria use several mechanisms for activating exogenous fatty acids, one of the most important of which is the conversion of exogenous fatty acids to acyl-CoA, which is then broken down by β-oxidation to produce acetyl-CoA (Kallscheuer et al, 2017).…”

Countering Gram-Negative Antibiotic Resistance: Recent Progress in Disrupting the Outer Membrane with Novel Therapeutics

“…The success of this screen illustrates how such strategies can be useful tools for the targeted discovery of compounds against an OM pathway. Indeed, an analogous strategy has also identified an inhibitor of the late LPS biosynthesis enzyme LpxK [85].…”