Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles

Abstract

Recent mutations in RND efflux pumps in clinical strains have further increased multidrug resistance. We show that R717L and R717Q substitutions (found in azithromycin-resistant Salmonella enterica spp.) in the Escherichia coli efflux pump AcrB dramatically increase macrolide, as well as fluoroquinolone, resistance. On the other hand, cells became more susceptible to novobiocin and β-lactam cloxacillin. We urge the control of, and adjustments to, treatments with antibiotics and the need for novel antibiotics and efflux pump inhibitors.

Keywords: AcrB; RND; antimicrobial resistance; fluoroquinolones; macrolides; multidrug resistance.

FIG 1

Location of Arg717 and the R717Q/L amino acid substitutions in AcrB-Ec. (a) Side view of the entire AcrB-Ec trimer. (b) A close-up view of the rectangle area highlighted in panel a. Two drug molecules are present in the proximal binding pocket (rifampicin and erythromycin). The Arg717 residue and the R717Q/L substitutions are located at the entrance of the pocket. (c) Close-up inwards view of the proximal binding pocket and Arg717 area. Rifampicin can be seen behind the Arg717 residue and the R717Q/L substitutions. CH1–4, Channel 1–4. Colors: red box, substitution area; yellow spheres, rifampicin; orange spheres, erythromycin; green sticks, Arg717; orange sticks, R717Q; red sticks, R717L. PDB accession codes: 4DX5 (for the main structure and the mutagenesis); 3AOC (for the erythromycin coordinates); 3AOD (for the rifampicin and the wild-type Arg717 side chain coordinates).

FIG 2

Growth ability of E. coli MG1655ΔacrB cells expressing wild-type, R717Q, and R717L mutant AcrB-Ec under several macrolide concentrations. Top lane, growth ability under erythromycin; middle lane, under clarithromycin; bottom lane, under azithromycin. Blue, yellow, red, and green indicate wild-type AcrB, R717Q mutant, R717L mutant, and vector only, respectively. The green arrow mark indicates the increase in growth ability of the mutant strains compared to the wild-type strain. ERY, erythromycin; CLR, clarithromycin; AZM, azithromycin. Experiments repeated twice provided similar results; shown is one of the results.

FIG 3

Growth ability of E. coli MG1655ΔacrB cells expressing wild-type, R717Q, and R717L mutant AcrB-Ec under cloxacillin and novobiocin. Top lane, growth ability under cloxacillin; bottom lane, under novobiocin. Blue, yellow, red, and green indicate wild-type AcrB, R717Q mutant, R717L mutant, and vector only, respectively. The red arrow mark indicates the decrease in growth ability of the mutant strains compared to the wild-type strain. CLX, cloxacillin; NOV, novobiocin. Experiments repeated twice provided similar results; shown is one of the results.

FIG 4

Kirby-Bauer disk diffusion susceptibility testing of E. coli MG1655ΔacrB cells expressing wild-type, R717Q, and R717L mutant AcrB-Ec under various antibiotics. (a) Grayscale images of knockout, wild-type, and mutant E. coli grown on Mueller-Hilton agar plates, supplemented with resazurin and arabinose. Disks with specific antibiotics of interest can be seen on all four plates, along with the growth inhibition zones around the disks. (b) Quantification of the inhibition zones. The vertical axis shows the inhibition diameter in mm. Growth of the cells up to the antibiotic disk is denoted as 0 mm. (a–b) Blue, yellow, red, and green indicate wild-type AcrB, R717Q mutant, R717L mutant, and vector only, respectively. KO, acrB knockout (vector only); WT, wild-type; ERY, erythromycin; CLR, clarithromycin; AZM, azithromycin; NOV, novobiocin; LVX, levofloxacin; OFX, ofloxacin; MXF, moxifloxacin; MIN, minocycline. Shown is one of the results; repeats gave similar results.

FIG 5

Comparison of the entrance area of the proximal binding pocket in the Access (or Loose [L]) monomer of AcrB-Ec for wild-type and the mutants. (a) Wild-type, (b) R717Q mutant, and (c) R717L mutant AcrB-Ec. (a–c) The residues of interest are depicted as pink sticks. The electrostatic surface is depicted from red to blue. The bold lines indicate the wild-type AcrB-Ec proximal binding pocket entrance area at the Arg717 location. In (b) and (c), the green highlighted area depicts the increased area for R717Q (Gln717) and R717L (Leu717) mutant AcrB-Ec, respectively. These images show the entrance of the proximal binding pocket, leading to the distal binding pocket in the background. PBP, proximal binding pocket; DBP, distal binding pocket. PDB accession code: 4DX5.