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

Richie, Daryl and Wang, Lisha and Chan, Helen and De Pascale, Gianfranco and Six, David and Wei, Jun-Rong and Dean, Charles (2018) A pathway-directed positive growth restoration assay to facilitate the discovery of lipid A and fatty acid biosynthesis inhibitors in Acinetobacter baumannii. PLoS ONE, 13 (3). ISSN 1932-6203

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 LPS 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 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 LPS 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 and optimization of compounds inhibiting known pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.

Item Type: Article
Date Deposited: 17 Mar 2018 00:45
Last Modified: 17 Mar 2018 00:45
URI: https://oak.novartis.com/id/eprint/34637

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