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. 2008 Sep 9;105(36):13586-91.
doi: 10.1073/pnas.0803391105. Epub 2008 Aug 29.

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

Hao Chen  1 , Jie Hu, Peng R Chen, Lefu Lan, Zigang Li, Leslie M Hicks, Aaron R Dinner, Chuan He
Affiliations

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

Hao Chen et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
P. aeruginosa MexR. (A) MexR is a transcriptional repressor of the mexAB–oprM multidrug efflux operon. Oxidation stress may serve as a signal to activate MexR. (B) Sequence and secondary structure of MexR with 3 Cys residues highlighted.
Fig. 2.
Fig. 2.
Dissociation of oxidized MexR from DNA. (A) EMSA of MexR with a DNA probe (50 nM) that contains MexR-binding sequence showing formation of a MexR–DNA complex. (B) Oxidation of MexR (1 μM per dimer) with CHP (0–125 μM) led to dissociation of the protein from DNA. Lane 1, 1 μM MexR formed a complex with DNA; lane 2, DNA probe alone; lanes 3–11, oxidation of MexR with varying concentrations of CHP for 60 min before being applied to the shift assay. (C) Cys alkylation blocks oxidation-induced dissociation of MexR (1 μM per dimer) from DNA (50 nM). Lane 1, MexR–DNA complex; lane 2, DNA alone; lanes 3 and 4, oxidized MexR by CHP (80 and 800 μM) did not bind DNA; lane 5, MexR, alkylated with 80 μM phenyl vinyl sulfonate (ALK1), still bound DNA; lanes 6 and 7, MexR, alkylated with phenyl vinyl sulfonate (80 μM) for 50 min before treating with CHP (80 and 800 μM) for 90 min, still bound DNA; lane 8, same as lane 5 except with iodoacetamide (ALK2) as the alkylator; lanes 9 and 10, same as lanes 6 and 7 except with ALK2.
Fig. 3.
Fig. 3.
Biochemical assays for MexR oxidation. (A) Quantification of free thiols in MexR in the reduced and oxidized protein. The reduced MexR contains three thiols per monomer. One equiv of thiol per monomer remained after treating MexR with 3 equiv of CHP for 1 h at room temperature. Both the C30S and C62S mutants showed 1 equiv of thiol remaining after the same CHP treatment; however, almost no thiol remained for the C138S mutant after it was treated with CHP. (B) EMSA (with 50 nM DNA) with various forms of MexR (0.5 μM). The wild-type (WT), C30S, C62S, and C138S all bound DNA when freshly purified. Treating these proteins with CHP (100 μM) or H2O2 (100 μM) for 40 min led to dissociation of the wild-type (lanes 6 and 10) and C138S mutant (lanes 9 and 13) from DNA, indicating that Cys-30 and Cye62 are involved in oxidation sensing. Lane 5 is control with DNA only.
Fig. 4.
Fig. 4.
Mass spectrometric mapping of disulfide bond in MexR and the simulated structure of oxidized MexR. (A) ESI-Q-TOF mass spectrum (m/z 200–1,000) of an unfractionated tryptic digestion. (Inset) The 3+ charge state (m/z 540–546) corresponding to the disulfide-containing peptide of interest (theoretical molecular mass, 1,624.73 Da). (B) MS/MS fragmentation of the 3+ charge state (m/z 542). (C) Graphical fragment map correlating the fragmentation ions to the sequence of the disulfide-containing peptide. The disulfide-linked cysteines are circled. (D) Structure of the reduced MexR dimer; the “open” CD dimer from PDB 1LNW (α4 and α4′ are the two DNA-binding helices). (E) Computationally predicted “closed” form with the disulfide bonded Cys-30–Cys-62 indicated.
Fig. 5.
Fig. 5.
Redox potential measurement for MexR. (A) Various concentrations of GSH and GSSG were incubated with 25 μM freshly purified MexR and C138S mutant protein at pH 7.4 and 37°C for 2 h. Reaction was stopped by adding excess iodoacetamide (19 mM). Protein samples were analyzed by nonreducing, denaturing SDS/PAGE. (B) Fitting of the titration data according to Reaction 1. Triangles (wild-type MexR) and circles (C138S mutant) correspond to experimental data, and the solid line is the theoretic fit. The redox potential was determined to be around −155 mV.
Fig. 6.
Fig. 6.
Plate sensitivity assay. Four P. aeruginosa strains were treated by CHP and different antibiotics. Row 1, wide-type PAO1 with pAK1900 (the empty plasmid); row 2, mexR mutant with pAK1900; row 3, mexR mutant complemented by mexR in pAK1900; row 4, mexR mutant complemented with mexRC30SC62S in pAK1900. The control plate had no antibiotics.
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