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

Abstract

The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain.

Importance: The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

FIG 1

Western blot of purified envelopes. Envelopes were purified from overnight cultures of a ΔacrAB strain containing either the empty vector plasmid pACYC184 (first lane) or plasmid clones expressing various AcrB proteins, as indicated. Detergent-solubilized envelope samples were analyzed by SDS-PAGE and electrotransferred to PVDF membranes. Membranes were blotted with primary antibodies against AcrB-MBP. LC, nonspecific band used as a gel loading control. AcrB levels were determined relative to those of LC and then normalized to the wild-type value of 1.

FIG 2

X-ray structure of AcrB (PDB entry 2GIF). Only the AcrB binding state protomer is shown. The positions of F610, F615, residues affected by suppressor alterations, and functionally important sites in AcrB are shown.

FIG 3

NPN efflux assays. Efflux of NPN from cells preloaded with this dye was initiated by adding 50 mM glucose. A rapid loss of fluorescence intensity indicates AcrAB-mediated efflux of NPN. The excitation and emission wavelengths were set at 340 nm and 410 nm, respectively. For clarity, data for AcrB suppressors mapping to the porter domain are shown in panel A, whereas those for suppressors mapping to the TM domain are shown in panel B. Slopes (m) and t_efflux50% values are shown in Table 5. a.u., arbitrary units.

FIG 4

Nitrocefin efflux assays. Cell preparation and assays were carried out as described by Nagano and Nikaido (39) and are described in Materials and Methods. R, Pearson correlation coefficient; h, Hill coefficient. R and h values are shown for strains displaying Michaelis-Menten kinetics and sigmoidal kinetics, respectively.