A cell-based infection assay identifies efflux pump modulators that reduce bacterial intracellular load.

Abigail L Reens,Jessica D Podoll,Corrella S Detweiler,Madeline E Edwards,David L Reens,Toni A Nagy,Edward W Yu,Chih-Chia Su,Amy L Crooks,Samuel I. Miller

doi:10.1371/journal.ppat.1007115

Abigail L Reens, Jessica D Podoll + Show 8 more

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https://doi.org/10.1371/journal.ppat.1007115

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Abstract

Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAβN (Phe-Arg β-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.

Highlights

Human pathogens have become increasingly resistant to clinical antibiotics
Bacteria evolved molecular machines called efflux pumps to export toxic chemicals, including antibiotics encountered in the environment
We describe an infection-based screen that identified chemicals that inhibit bacterial efflux pump activity and show that these compounds bind to and block the activity of bacterial efflux pumps

Summary

Introduction

Human pathogens have become increasingly resistant to clinical antibiotics. Gram-negative bacterial pathogens are problematic because their outer membranes are impermeable to many chemicals, and because many compounds that do enter the periplasm or cross the cellular membrane are immediately exported by efflux pumps. Multi-drug resistant (MDR) bacteria typically have increased gene copy number and/or production of efflux pumps, features demonstrated to contribute to the failure of clinical antibiotic treatment [1]. For these reasons, compounds that reduce efflux pump activity (efflux pump modulators, EPMs) are under investigation for their potential use in re-sensitizing MDR pathogens to existing antibiotics [2]. PAβN binds AcrB, the main component of the efflux system AcrAB-TolC, a member of the RND (resistance-nodulation-cell division) family of pumps This compound was not developed as an antibiotic because medicinal chemistry could not separate EPM activity from unfavorable pharmacokinetics and toxicology, possibly reflecting off-target effects [5,6]. We identified three compounds that have activity as EPMs using a different approach—an in-cell screen for small molecules that prevent the replication of the Gram-negative pathogen Salmonella enterica (Salmonella) in mammalian cells

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