CpxR Activates MexAB-OprM Efflux Pump Expression and Enhances Antibiotic Resistance in Both Laboratory and Clinical nalB-Type Isolates of Pseudomonas aeruginosa

Abstract

Resistance-Nodulation-Division (RND) efflux pumps are responsible for multidrug resistance in Pseudomonas aeruginosa. In this study, we demonstrate that CpxR, previously identified as a regulator of the cell envelope stress response in Escherichia coli, is directly involved in activation of expression of RND efflux pump MexAB-OprM in P. aeruginosa. A conserved CpxR binding site was identified upstream of the mexA promoter in all genome-sequenced P. aeruginosa strains. CpxR is required to enhance mexAB-oprM expression and drug resistance, in the absence of repressor MexR, in P. aeruginosa strains PA14. As defective mexR is a genetic trait associated with the clinical emergence of nalB-type multidrug resistance in P. aeruginosa during antibiotic treatment, we investigated the involvement of CpxR in regulating multidrug resistance among resistant isolates generated in the laboratory via antibiotic treatment and collected in clinical settings. CpxR is required to activate expression of mexAB-oprM and enhances drug resistance, in the absence or presence of MexR, in ofloxacin-cefsulodin-resistant isolates generated in the laboratory. Furthermore, CpxR was also important in the mexR-defective clinical isolates. The newly identified regulatory linkage between CpxR and the MexAB-OprM efflux pump highlights the presence of a complex regulatory network modulating multidrug resistance in P. aeruginosa.

Fig 1. Comparative genomic analysis of conserved DNA motifs.

(A) A well-conserved DNA motif exists in the promoter regions of orthologous muxABC-opmB operons in Pseudomonas species. The sequence logo for the conserved DNA motif reflects position-specific probability matrixes; high probability (≥ 70%) nucleotides are marked in grey in the alignment. The DNA motif contains a well-conserved CpxR binding site. The number in the blanket is the distance between the DNA motif and the ATG start codon of each gene locus. (B) DNA sequence of the mexR-mexA intergenic region. The ATG start codons of mexA and mexR are under solid arrows, indicating the directions of the coding sequences. The -35 and -10 regions of the distal and proximal promoters of mexA are underlined [12, 40]. The transcriptional start sites of the distal and proximal promoters of mexA are indicated by bent arrows [12, 40]. The putative CpxR binding site is shaded, whereas the two MexR binding sites overlapping the -35/-10 region of the distal promoter are indicated in lower-case letters [40, 41]. A NalD binding site overlapping the -35/-10 region of the proximal promoter is indicated in italic lower-case letters [12]. The nucleotide substitutions for the mutated mexA promoters (mexAp_M1 and mexAp_M3) are paired with a short vertical line. The downstream deletion boundary of the distal-only mexA promoter (mexAp_M2) is indicated with a long vertical line. (C) A well-conserved CpxR binding site exists in the promoter region of each cpxP and muxA orthologue, while a well-conserved NalD binding site exists in the promoter region of each mexA orthologue. In contrast, the conserved CpxR and MexR binding site on the mexA promoter is unique to P. aeruginosa. (D) Components that might be involved in regulating mexA orthologue (in blue) expression in Pseudomonas species. The orthologous loci of cpxR and nalD among different Pseudomonas species are marked in green and yellow, respectively. The orthologous nalD locus is separated and replaced by the mexR locus in P. aeruginosa, but divergently linked to the orthologous mexA locus in the genomes of other Pseudomonas species.

Fig 2. Direct binding of CpxR to the promoter region of mexA in vitro.

DNase I footprinting assays of the mexA promoter DNA fragment were performed in the absence (A) and presence (B) of purified CpxR. The FAM-labelled 322-bp DNA fragments (50 nM) pre-incubated in the absence or presence of 1.5 μM phosphorylated CpxR were subjected to DNase I digestion and fragment length analysis. The fluorescence signal of the labelled DNA fragments is plotted against the sequence of the fragment. The protected region bound by CpxR is shown with the conserved binding motif in red.

Fig 3. CpxR-mediated up-regulation of mexAB-oprM expression in mexR-deleted P. aeruginosa PA14.

(A) The expression levels of mexAp::lacZ reporter as measured by β-galactosidase assay in the PA14, PA14ΔcpxR, PA14ΔmexR, and PA14ΔcpxRΔmexR strains. Each bar represents the mean ± SD of two independent experiments (**, p < 0.01). (B) Relative mexB transcript levels determined by quantitative real-time PCR in the PA14, PA14ΔcpxR, PA14ΔmexR, and PA14ΔcpxRΔmexR strains. Data are expressed relative to the quantity of mexB mRNA in the wild-type PA14 strain. Each bar represents the mean ± SD of the relative quantity in three independent experiments (*, p < 0.05). (C) Relative MexA protein levels were determined by western blotting in the PA14, PA14ΔcpxR, PA14ΔmexR, and PA14ΔcpxRΔmexR strains. The intensity of each band was quantified. The results are expressed relative to the quantity of MexA in the wild-type PA14 strain. Each bar represents the mean ± SD of the relative quantity in three independent experiments (*, p < 0.05). (D) A representative western blot image of MexA protein in the PA14, PA14ΔcpxR, PA14ΔmexR, and PA14ΔcpxRΔmexR strains. The membrane proteins (10 μg per lane) were separated by 10% SDS-PAGE and immunoblotted with anti-MexA polyclonal antibodies.