Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli

Abstract

The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine ␤-naphthylamide (PA␤N) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PA␤N and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PA␤N, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PA␤N. Strong action of PA␤N in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PA␤N acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PA␤N acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PA␤N in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PA␤N on membrane integrity are compounded in cells unable to extrude PA␤N. IMPORTANCEThe increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds. Multidrug resistance among human bacterial pathogens remains a grave social concern. Numerous strategies have been proposed to curtail the rampant increase in multidrug resistance among human pathogens, ranging from the effective integration of pharmacokinetic and pharmacodynamic parameters and implementation of antimicrobial stewardship (1) to the development of novel antibiotics based on either an existing or a novel chemical scaffold, exploitation of new cellular targets, and directly tackling the cellular mechanisms that confer multidrug resistance (2). Efflux of antibiotics from the cell is one of the common mechanisms of antibiotic resistance in bacteria, with resistance developing when the rate of drug efflux across the membrane exceeds that of drug influx (3).Bacterial genomes encode several membrane-bound multidrug efflux systems (4, 5). These systems are usually under t...

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

“…For this, we first employed NPN, a small fluorescent compound that is a substrate of AcrB (37,46). NPN fluoresces strongly when partitioned in the lipophilic environment of the membrane but only weakly when present in the aqueous environment of the medium (32,47). NPN was loaded into cells in which AcrB was transiently inactivated by treatment with the protonophore CCCP.…”

“…Efflux of NPN in live bacterial cells was carried out essentially as described by Lomovskaya et al (37), with some modifications (32). Briefly, overnight cultures were centrifuged, and pellets were washed with potassium phosphate buffer (20 mM KPO 4 , pH 7.0) containing 1 mM MgCl 2 and then centrifuged again.…”

“…At the 100-s time point, efflux of NPN was initiated by adding 50 mM (final concentration) glucose, and changes in fluorescence intensity were measured for 100 s. Slopes [m ϭ (y 2 Ϫ y 1 )/(x 2 Ϫ x 1 )] resulting from the decrease in NPN fluorescence were calculated, with values expressed in fluorescence intensities per second (FI/s). Using ⌬acrAB cells, which allow maximal accumulation of NPN inside the cell, it was determined that an NPN concentration as high as 20 M does not lead to self-quenching of fluorescence (32).…”

“…The bacterial strains used in the study were derived from RAM2370 (MC4100 ⌬ara ⌬acrAB:: scar) (9,32). Luria broth (LB) was prepared from LB Broth EZMix powder (Lennox).…”

Two Outer Membrane Proteins Contribute to Caulobacter crescentus Cellular Fitness by Preventing Intracellular S-Layer Protein Accumulation

“…Consequently, an increase in the amount of LPS in the outer membrane during this rearrangement could lead to a change in outer membrane permeability and increased diffusivity of nonpolar compounds, such as antibiotics, across the outer membrane (53). To test whether cells deficient in RsaF a and RsaF b exhibited altered membrane permeability, we conducted the nitrocefin hydrolysis assay developed by Misra et al (54). Nitrocefin can diffuse across the outer membrane and into the periplasm, where ␤-lactamase enzymes can cleave a ␤-lactam ring on nitrocefin, causing a color change of the assay solution.…”