Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Abstract

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a wellknown inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.RND transporters | efflux inhibitors | Phe-Arg-β-naphthylamide | molecular dynamics simulations T he emergence and spread of antibiotic-resistant pathogenic bacteria, especially those of multidrug-resistant or even panresistant Gram-negative bacteria, is a major problem (1). Multidrug efflux pumps, especially those of the resistance-nodulation division (RND) family, contribute strongly to this type of resistance (2). AcrB of Escherichia coli has very wide substrate specificity (3) and its structure and mechanism have been studied extensively as a prototype RND efflux pump (4, 5). AcrB enhances the intrinsic resistance of E. coli, especially to large or lipophilic antibiotics (3), and makes the cell more resistant when overproduced (6). Because of this role, inhibitors of AcrB and its relatives are important not only in basic research but also possibly in clinical medicine, as they could reduce the resistance level of pathogens. The first such inhibitor was Phe-Arg β-naphthylamide (PAβN), reported by Lomovskaya et al. in 2001 (7). These researchers also showed that a PAβN analog, Ala β-naphthylamide, is a good substrate for the AcrB homolog MexB of Pseudomonas aeruginosa, by examining its intracellular accumulation followed by enzymatic hydrolysis that generated the fluorescent naphthylamine. The researchers suggested that PAβN is also a substrate of the AcrB/MexB pump, because pump overproducer...

Effect of site-directed mutations in multidrug efflux pump AcrB examined by quantitative efflux assays

“…These different binding behaviors of substrates are consistent with the idea that substrates tend to bind to one of the two sub-areas of the distal binding pocket. More recently, some of the aminoacyl-β-naphthylamides, including Ala-Naph, was found to bind mostly to the hydrophobic pocket or the cave [14]. We therefore tested quantitatively the effect of mutations in the distal binding pocket on the efflux of both a groove binder, nitrocefin, and a cave binder, Ala-Naph.…”

“…For the determination of Ala-Naph efflux, ΔpepN :: kan mutation was first transduced into RAM121 from JW0915-1 in Keio collection (obtained from E. coli genetic stock center, Yale University), followed by the transduction of ΔacrAB :: spc [14]. RAM121 ΔpepN :: kan ΔacrAB :: spc was then transformed with pMAL-PepN [14] and the transformants were selected on 100 μg/ml ampicillin plates at 37°C.…”

“…RAM121 ΔpepN :: kan ΔacrAB :: spc was then transformed with pMAL-PepN [14] and the transformants were selected on 100 μg/ml ampicillin plates at 37°C. This transformant was then transformed with pACYC184 acrAB (with or without a mutation within AcrB) followed by selection on LB plates containing both ampicillin (100 μg/ml) and chloramphenicol (35 μg/ml).…”

“…The cell suspension was then diluted (1:8) in the same buffer. The Ala-Naph efflux assay was carried out as described previously [14]. The external concentrations of Ala-Naph varied usually up to 1.0 mM.…”

“…This approach, however, is very crude, because MICs are the results of interactions of many processes, including influx, efflux, drug inactivation, and drug binding, and they are unlikely to be the direct indicator of the efflux process alone. We have, however, succeeded in determining the kinetics of the efflux of two types of substrates, β-lactams [12, 13] and aminoacyl-β-naphthylamides [14]. We have generated AcrB mutants in which various residues in the substrate-binding cavities were converted to alanine, and we have directly determined the effect of these mutations on efflux kinetics, by using representatives of these two types of substrates, nitrocefin (a cephalosporin) and Ala-β-naphthylamide or Ala-Naph (an aminoacyl-β-naphthylamide).…”