Mechanism of plasmid-mediated quinolone resistance

Abstract

Quinolones are potent antibacterial agents that specifically target bacterial DNA gyrase and topoisomerase IV. Widespread use of these agents has contributed to the rise of bacterial quinolone resistance. Previous studies have shown that quinolone resistance arises by mutations in chromosomal genes. Recently, a multiresistance plasmid was discovered that encodes transferable resistance to quinolones. We have cloned the plasmid-quinolone resistance gene, termed qnr, and found it in an integron-like environment upstream from qacE Delta 1 and sulI. The gene product Qnr was a 218-aa protein belonging to the pentapeptide repeat family and shared sequence homology with the immunity protein McbG, which is thought to protect DNA gyrase from the action of microcin B17. Qnr had pentapeptide repeat domains of 11 and 28 tandem copies, separated by a single glycine with a consensus sequence of A/C D/N L/F X X. Because the primary target of quinolones is DNA gyrase in Gram-negative strains, we tested the ability of Qnr to reverse the inhibition of gyrase activity by quinolones. Purified Qnr-His(6) protected Escherichia coli DNA gyrase from inhibition by ciprofloxacin. Gyrase protection was proportional to the concentration of Qnr-His(6) and inversely proportional to the concentration of ciprofloxacin. The protective activity of Qnr-His(6) was lost by boiling the protein and involved neither quinolone inactivation nor independent gyrase activity. Protection of topoisomerase IV, a secondary target of quinolone action in E. coli, was not evident. How Qnr protects DNA gyrase and the prevalence of this resistance mechanism in clinical isolates remains to be determined.

Figure 1

Map of the EcoRI fragment of pMG252 encoding quinolone resistance. The qnr gene is immediately upstream from qacEΔ1, which encodes resistance to quaternary ammonium compounds, and sulI, an unexpressed gene for sulfonamide resistance. Two copies of orf513, found in integrons In6 and In7, surround these genes. Restriction sites mentioned in the text are also shown.

Figure 2

The amino acid sequence of Qnr written to emphasize the pentapeptide repeating unit with a consensus sequence of A/C D/N L/F X X. The protein can be divided into domains of 11 and 28 units connected by a single glycine. The conserved amino acid residues are highlighted.

Figure 3

Qnr reversal of DNA gyrase inhibition. Reaction mixtures contained no enzyme (lane 1) or 0.93 nM DNA gyrase (lanes 2–6) and ciprofloxacin at concentration 1.5 μM (0.5 μg/ml) (lanes 3–6). The assays were performed in the presence of 2.01 μM Qnr from cells induced at a final concentration of 1 mM isopropyl 1-thio-β-d-galactopyranoside (IPTG) (lane 4) or 1.2 μM Qnr from uninduced cells (lane 5), and from IPTG-induced cells transformed with the expression vector pQE60 alone (lane 6).

Figure 4

Stoichiometry of Qnr protection. Reactions contained no enzyme (lane 1) or 0.93 nM DNA gyrase (lanes 2–10) and 1.5 μM ciprofloxacin (lanes 3–10). The assays were performed in the presence of Qnr at concentrations of 2,170 nM (lane 4), 725 nM (lane 5), 242 nM (lane 6), 81 nM (lane 7), 27 nM (lane 8), 9 nM (lane 9), and 3 nM (lane 10).

Figure 5

Dependence of Qnr protection on ciprofloxacin concentration. Reactions contained no enzyme (lane 1) or 0.93 nM DNA gyrase (lanes 2–10) and Qnr at 2.01 μM (lanes 4, 6, 8, 10). The assays were performed in the presence of ciprofloxacin at 0.75 μM (lanes 3 and 4), 1.5 μM (lanes 5 and 6), 3.0 μM (lanes 7 and 8), and 6.0 μM (lanes 9 and 10).

Figure 6

Lack of supercoiling activity. Reactions contained no enzyme (lane 1) or DNA gyrase at concentrations of 0.93 nM (lanes 2 and 3), 0.62 nM (lanes 4 and 5), or 0.46 nM (lanes 6 and 7). The assays were performed in the presence of Qnr at 3.12 μM (lanes 3, 5, and 7).

Figure 7

Failure of Qnr to protect topoisomerase IV. Reactions contained no enzyme (lane 1) or 20.3 nM topoisomerase IV (lanes 2–10) and 24.1 μM ciprofloxacin (lanes 3–10). Qnr was present at 6.5 μM (lane 4), 3.4 μM (lane 5), 1.7 μM (lane 6), 0.58 μM (lane 7), 0.19 μM (lane 8), or 0.06 μM (lane 9), and BSA was added at 3.2 μM (lane 10).

Figure 8

clustal w alignment of Qnr, McbG (Swissprot P05530), and MfpA (24).