Identifying Modulators of the Post-Antibiotic Effect

Abstract

The post-antibiotic effect (PAE) is the delay in bacterial regrowth following antibiotic removal. It has important implications for dosing regimens since drugs that have extended activity following their elimination can be dosed less frequently, widening the therapeutic window. While the PAE has been associated with target vulnerability and the rate of target turnover, little is known about the genetic components that modulate the PAE. Here, we developed a high-throughput assay to screen the Escherichia coli Keio collection of [~]4000 deletion strains, identifying genes that enhance the PAE for CHIR-090, an inhibitor of UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC). This screen revealed approximately 400 gene knockouts that enhanced the PAE of CHIR-090. The list of PAE enhancers was enriched for genes involved in transmembrane transport and outer membrane synthesis. Notably, deletion of the rfaE gene, which is involved in lipopolysaccharide (LPS) biosynthesis, increased the PAE of the LpxC inhibitors CHIR-090 and LPC-058 by 2 h and 3 h, respectively. Consistent with this phenotype, co-treatment of wild-type E. coli with an RfaE inhibitor increased the PAE of CHIR-090 or LPC-058 by 1 h. To probe the mechanism of this interaction, we measured the rate of LpxC turnover and found that knocking out rfaE reduced its half-life by 2-fold, suggesting that disrupting RfaE increases the stability of LpxC, increasing target vulnerability and enhancing the PAE of LpxC inhibitors. SIGNIFICANCEAntibiotics are generally dosed at very high levels leading to unwanted side effects and non-compliance, which in turn results in the emergence of drug-resistant bacterial infections. The goal of this work was to develop strategies that will enable antibiotics to be dosed at lower levels, thereby improving safety and compliance. In the present work, we have screened 4,000 strains of Escherichia coli to identify compounds that result in an increase in the post-antibiotic effect (PAE), which is the delay in bacterial regrowth following antibiotic exposure and removal. Drugs that cause a PAE are dosed less frequently, and the method we describe will provide a new approach to developing safer drugs.