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The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism.

ABSTRACT: MexR is a MarR family protein that negatively regulates multidrug efflux systems in the human pathogen Pseudomonas aeruginosa. The mechanism of MexR-regulated antibiotic resistance has never been elucidated in the past. We present here that two Cys residues in MexR are redox-active. They form intermonomer disulfide bonds in MexR dimer with a redox potential of -155 mV. This MexR oxidation leads to its dissociation from promoter DNA, derepression of the mexAB-oprM drug efflux operon, and increased antibiotic resistance of P. aeruginosa. We show computationally that the formation of disulfide bonds is consistent with a conformation change that prevents the oxidized MexR from binding to DNA. Collectively, the results reveal that MexR is a redox regulator that senses peroxide stress to mediate antibiotic resistance in P. aeruginosa.

SUBMITTER: Chen H

PROVIDER: S-EPMC2533233 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism.

Chen Hao H Hu Jie J Chen Peng R PR Lan Lefu L Li Zigang Z Hicks Leslie M LM Dinner Aaron R AR He Chuan C

Proceedings of the National Academy of Sciences of the United States of America 20080829 36

MexR is a MarR family protein that negatively regulates multidrug efflux systems in the human pathogen Pseudomonas aeruginosa. The mechanism of MexR-regulated antibiotic resistance has never been elucidated in the past. We present here that two Cys residues in MexR are redox-active. They form intermonomer disulfide bonds in MexR dimer with a redox potential of -155 mV. This MexR oxidation leads to its dissociation from promoter DNA, derepression of the mexAB-oprM drug efflux operon, and increase ...[more]

PMID: 18757728

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Project description:The region upstream of the multiple antibiotic resistance efflux operon mexA-mexB-oprM in Pseudomonas aeruginosa was sequenced, and a gene, mexR, was identified. The predicted MexR product contains 147 amino acids with a molecular mass of 16,964 Da, which is consistent with the observed size of the overexpressed mexR gene product. MexR was homologous to MarR, the repressor of MarA-dependent multidrug resistance in Escherichia coli, and other repressors of the MarR family. A mexR knockout mutant showed a twofold increase in expression of both plasmid-borne and chromosomal mexA-reporter gene fusions compared with the MexR+ parent strain, indicating that the mexR gene product negatively regulates expression of the mexA-mexB-oprM operon. Furthermore, the cloned mexR gene product reduced expression of a plasmid-borne mexA-lacZ fusion in E. coli, indicating that MexR represses mexA-mexB-oprM expression directly. Consistent with the increased expression of the efflux operon in the mexR mutant, the mutant showed an increase (relative to its MexR+ parent) in resistance to several antimicrobial agents. Expression of a mexR-lacZ fusion increased threefold in a mexR knockout mutant, indicating that mexR is negatively autoregulated. OCR1, a nalB multidrug-resistant mutant which overproduces OprM, exhibited a greater than sevenfold increase in expression of a chromosomal mexA-phoA fusion compared with its parent. Introduction of a mexR knockout mutation in strain OCR1 eliminated this increase in efflux gene expression and, as expected, increased the susceptibility of the strain to a variety of antibiotics. The nucleotide sequences of the mexR genes of OCR1 and its parental strain revealed a single base substitution in the former which would cause a predicted substitution of Trp for Arg at position 69 of its mexR product. These data suggest that MexR possesses both repressor and activator function in vivo, the activator form being favored in nalB multidrug-resistant strains.

Project description:Pseudomonas aeruginosa has different resistant mechanisms including the constitutive MexAB-OprM efflux pump. Single nucleotide polymorphisms (SNPs) in the mexR, nalC, and nalD repressors of this efflux pump can contribute to antimicrobial resistance; however, it is unknown whether these changes are mainly related to genetic lineages or environmental pressure. This study identifies SNPs in the mexR, nalC, and nalD genes in clinical and environmental isolates of P. aeruginosa (including high-risk clones). Ninety-one P. aeruginosa strains were classified according to their resistance to antibiotics, typified by multilocus sequencing, and mexR, nalC, and nalD genes sequenced for SNPs identification. The mexAB-oprM transcript expression was determined. The 96.7% of the strains were classified as multidrug resistant. Eight strains produced serine carbapenemases, and 11 strains metallo-β-lactamases. Twenty-three new STs and high-risk clones ST111 and ST233 were identified. SNPs in the mexR, nalC, and nalD genes revealed 27 different haplotypes (patterns). Sixty-two mutational changes were identified, 13 non-synonymous. Haplotype 1 was the most frequent (n = 40), and mainly identified in strains ST1725 (33/40), with 57.5% pan drug resistant strains, 36.5% extensive drug resistant and two strains exhibiting serin-carbapenemases. Haplotype 12 (n = 9) was identified in ST233 and phylogenetically related STs, with 100% of the strains exhibiting XDR and 90% producing metallo-β-lactamases. Haplotype 5 was highly associated with XDR and related to dead when compared to ST1725 and ST233 (RRR 23.34; p = 0.009 and RRR 32.01; p = 0.025). A significant relationship between the mexR-nalC-nalD haplotypes and phylogenetically related STs was observed, suggesting mutational changes in these repressors are highly maintained within genetic lineages. In addition, phylogenetically related STs showed similar resistant profiles; however, the resistance was (likely or partly) attributed to the MexAB-OprM efflux pump in 56% of the strains (only 45.05% showed mexA overtranscription), in the remaining strains the resistance could be attributed to carbapenemases or mechanisms including other pumps, since same SNPs in the repressor genes gave rise to different resistance profiles.

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The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism.

Publications

The Pseudomonas aeruginosa multidrug efflux regulator MexR uses an oxidation-sensing mechanism.

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