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

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 MexEFOprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

Cinnamaldehyde Induces Expression of Efflux Pumps and Multidrug Resistance in Pseudomonas aeruginosa

“…For example, membrane-damaging agents, such as chlorhexidine (20) and benzalkonium chloride (43), are known inducers of operon mexCD-oprJ expression. As demonstrated previously in our laboratory, the aldehyde group of glyoxal or methylglyoxal strongly activates the production of MexEF-OprN and MexXY/OprM in treated bacteria (23). In agreement with these data, the present study shows that the conversion of the aldehyde group to a hydroxyl group abolishes the capacity of CNA to upregulate MexCD-OprJ, MexEF-OprN, and/or MexXY/ OprM.…”

“…Sequencing experiments confirmed the deletion of 5,238 bp in gene mexAB, 3,750 bp in gene mexXY, and 5,705 bp in operon mexCD-oprJ. A mutant lacking the entire operon mexEF-oprN (PA14ΔEFN) was already available in the laboratory (23). Double (PA14ΔAB/EFN, PA14ΔEFN/XY, and PA14ΔXY/CDJ), triple (PA14ΔAB/EFN/XY), and quadruple (PA14ΔAB/EFN/XY/CDJ) mutants defective in several Mex pumps were successively obtained by using the same approach as described above (Table S1).…”

“…Reminiscent of the action of some other essential oils (13,14), we found that P. aeruginosa adapts to relatively elevated CNA concentrations by upregulating several Mex pumps, of which MexAB-OprM is essential for its survival. Such a multipump activation likely results from a global response to the stress generated by CNA (23). The energy cost of such a high efflux activity might explain the transient growth arrest observed during the first hour of CNA exposure.…”

“…While it has become clear that mex genes are overexpressed under such harsh conditions (18)(19)(20)(21)(22), the impact of this increased efflux activity on antibiotic resistance has remained unexplored. Recently, we found that MexEF-OprN production is strongly augmented when P. aeruginosa is submitted to electrophilic stress by aldehydes, such as glyoxal, methylglyoxal, and cinnamaldehyde (CNA) (23). However, the intrinsic resistance of the microorganism to CNA appeared to be only partially dependent upon MexEF-OprN, thereby suggesting that other efflux systems also contribute to the protection against this compound (23).…”

Adaptive laboratory evolution restores solvent tolerance in plasmid-curedPseudomonas putidaS12; a molecular analysis

“…Indel mutations were observed in a hitherto uncharacterized AraC-family transcriptional regulator (Afr), causing it to be truncated in the ALE-derived strains. In P.putida KT2440, a homolog of Afr encoded by PP1395 (100% identity, 100% coverage) was found to be responsible for a decrease in glycerol uptake [39], while in P. aeruginosa PA14, Afr homolog (63% identity, 88% coverage) encoded by PA14_38040 (PA2074 in strain PAO1) was reported to regulate the expression of RND efflux pump MexEF-OprN [40]. Further characterization of Afr is underway.…”