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Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas Aeruginosa Through a Novel Transcriptional Regulator, CmrA

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Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas Aeruginosa Through a Novel Transcriptional Regulator, CmrA

Paulo Juarez et al. Antimicrob Agents Chemother.

Abstract

The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

Keywords: CmrA; MexEF-OprN; Pseudomonas aeruginosa; efflux; efflux pumps; electrophilic stress.

Figures

FIG 1
FIG 1
RaptorX prediction of CmrA structure. A three-dimensional structure of the regulator CmrA was modeled using the RaptorX Web server (http://raptorx.uchicago.edu/). The putative N-terminal ligand-binding domain (from amino acid position 40 to 191) was predicted based on ToxT from Vibrio cholerae (PDB 3GBG; P = 5.96e−4), while the putative C-terminal DNA-binding domain (from 198 to 310) is based on AdpA from Streptomyces griseus (PDB 3W6V; P = 3.45e−5). The amino acid substitutions found in PJ mutants are highlighted by blue spots.
FIG 2
FIG 2
Genetic environment of gene cmrA. (A) Gene annotations are those available in GenBank for strain PA14 (RefSeq accession number NC_008463.1). Homologs in strain PAO1 are indicated in brackets. The DNA sequence upstream from cmrA is shown below the schematic. Different positions were assigned to the start codon of cmrA in genomic maps of PA14 (PA14_38040, 933 bp; boldface and underlined) and PAO1 (PA2047, 990 bp; underlined). Two putative overlapping RpoN and RpoS binding motifs are highlighted in gray. The transcription start sites (TSS, +1) of cmrA and PA2048 were mapped by 5′-RACE at −38 bp and −315 bp, respectively. The 5′ UTR of PA2048 contains a 111-bp region of unknown function, bordered by two 10-bp inverted repeats (IR-N91). (B) Complementation experiments in mutant PA14ΔcmrA were carried out to clarify the role of the σ factor binding sites in cmrA expression. A full-length DNA fragment from mutant PJ01, carrying cmrA and the RpoN/RpoS binding sites (−152 bp upstream from the TSS), conferred an nfxC2 resistance phenotype on PA14ΔcmrA, while a short version lacking the RpoN/RpoS binding sites (−68 bp) did not. The whole sequence of cmrA and its 5′ UTR is accessible through the GenBank database (accession number KX274690).
FIG 3
FIG 3
Response of P. aeruginosa to electrophilic stress. (A) The bioluminescence of strain PA14::PA2048-lux was monitored at defined time points after exposure to increasing concentrations of glyoxal (GO) and is expressed as the ratio of relative light units (RLU) to bacterial density (A600). Nontreated bacteria were used as the control (CTRL). Results are mean values ± standard deviations from three independent experiments. (B) Expression levels of genes mexE and PA2048 were determined by RT-qPCR in strain PA14 exposed to 200 μg ml−1 GO and compared with those of a nontreated control (CTRL). Results are mean values of four determinations from two independent experiments. (C) Induction of pump MexEF-OprN with glyoxal was assessed by a double-disk antagonism test using strain PA14 and negative-control strain PA14ΔmexEF-oprN. Paper disks were loaded with 8,000 μg glyoxal (GO), 5 μg ciprofloxacin (CIP), 1,000 μg chloramphenicol (CHL), or 240 μg trimethoprim (TMP). Antagonism is visible between GO and all the tested MexEF-OprN substrates in strain PA14 (left) but not in mutant PA14ΔmexEF-oprN (right).
FIG 4
FIG 4
Bactericidal activity of cinnamaldehyde on P. aeruginosa. Bioluminescent strain PA14-lux and its derived mutants PA14-luxcmrA and PA14-luxmexEF-oprN were cultured to mid-log phase and then challenged with 1,000 μg ml−1 cinnamaldehyde. Bioluminescence (RLU) was recorded every 30 min and used as an indicator of cell survival. RLU values are mean values ± standard deviations from three independent experiments. A bioluminescence threshold was established with sterile MHB (dotted line).
FIG 5
FIG 5
Schematic representation of activation pathways of MexEF-OprN in P. aeruginosa. In wild-type strains (top left), such as strain PA14, regulators MexT and CmrA remain quiescent because of redox homeostasis. In so-called nfxC mutants (top right), mutational alteration of putative quinone oxidoreductase MexS is thought to result in intracellular accumulation of some redox-active MexS substrate(s). Redox-dependent oligomerization of MexT then triggers production of the pump MexEF-OprN and active efflux of still-undetermined endogenous products. In nfxC2 mutants (bottom left), regulator CmrA is activated as a result of gain-of-function mutations in gene cmrA. Among the 11 genes positively regulated by CmrA, PA2048 codes for a putative quinol monooxygenase. Concomitant activation of PA2048 and MexS is assumed to generate oxidized metabolites, the accumulation of which would modify the cellular redox state. As in MexS-deficient mutants, these changes upregulate the production of MexEF-OprN via MexT. Finally, upon electrophilic stress (bottom right), reactive electrophilic species (RES), such as glyoxal and methylglyoxal, induce the glyoxalase detoxification system (GloA2 and GloA3) and CmrA-dependent expression of MexEF-OprN. We propose that P. aeruginosa uses the efflux pump MexEF-OprN as a defense mechanism in response to toxic electrophilic stressors encountered in its environment.

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