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

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.

A macrophage-based screen identifies antibacterial compounds selective for intracellular Salmonella Typhimurium

“…However, others have shown that TolC is required, and therefore active, for intracellular growth of S . Tm 35,36 . We therefore reasoned that OM permeability, and not reduced efflux, contributes to metergoline activity in macrophages.…”

“…Intracellular pathogens often occupy niches that are difficult to recapitulate in vitro; thus, we and others have developed fluorescence, luminescence, and colorimetric-based screening assays with readouts that approximate bacterial viability when compared to traditional cfu counting methods 36,53–56 . Here we describe a macrophage-based chemical screen specific for intracellular S .…”

Genetic variation in the MacAB-TolC efflux pump influences pathogenesis of invasive Salmonella isolates from Africa

“…Furthermore, macAB is important for S . Typhimurium replication in macrophages [ 38 , 56 ], a niche where PhoP/Q signaling is active and important for Salmonella survival [ 55 , 61 ]. In addition to the different Salmonella isolates used across studies, our results suggesting PhoP promotes macAB transcription may arise from differences in experimental conditions, the importance of regulatory kinetics such as PhoP turnover [ 38 ], changes in ribosomal RNA levels in response to low magnesium [ 84 ], or coordinated activity with other transcriptional regulators [ 59 ].…”

“…Previous studies have shown that S. Typhimurium ST19 strains 14028S and SL1344 with deletions of macAB replicate poorly within mouse macrophages [38,56]. We infected the murine RAW264.7 macrophage cell line with a range of our marker-less macAB mutants to assess Salmonella intracellular replication.…”

A macrophage-based screen identifies antibacterial compounds selective for intracellularSalmonellaTyphimurium

“…Intracellular pathogens often occupy niches that are difficult to recapitulate in vitro; thus, we and others have developed fluorescence, luminescence, and colorimetric-based screening assays with readouts that approximate bacterial viability over traditional cfu counting methods 44-48 . Here we describe a macrophage-based chemical screen specific for intracellular S .…”