An EmrB multidrug efflux pump in Burkholderia thailandensis with unexpected roles in antibiotic resistance

Abstract

The antibiotic trimethoprim is frequently used to manage Burkholderia infections, and members of the resistance-nodulation-division (RND) family of efflux pumps have been implicated in multidrug resistance of this species complex. We show here that a member of the distinct Escherichia coli multidrug resistance B (EmrB) family is a primary exporter of trimethoprim in Burkholderia thailandensis, as evidenced by increased trimethoprim sensitivity after inactivation of emrB, the gene that encodes EmrB. We also found that the emrB gene is up-regulated following the addition of gentamicin and that this up-regulation is due to repression of the gene encoding OstR, a member of the multiple antibiotic resistance regulator (MarR) family. The addition of the oxidants H₂O₂ and CuCl₂ to B. thailandensis cultures resulted in OstR-dependent differential emrB expression, as determined by qRT-PCR analysis. Specifically, OstR functions as a rheostat that optimizes emrB expression under oxidizing conditions, and it senses oxidants by a unique mechanism involving two vicinal cysteines and one distant cysteine (Cys³, Cys⁴, and Cys¹⁶⁹) per monomer. Paradoxically, emrB inactivation increased resistance of B. thailandensis to tetracycline, a phenomenon that correlated with up-regulation of an RND efflux pump. These observations highlight the intricate mechanisms by which expression of genes that encode efflux pumps is optimized depending on cellular concentrations of antibiotics and oxidants.

Keywords: EmrB; MarR; antibiotic; bacterial virulence; efflux pump; gene regulation; multidrug resistance; multidrug transporter; oxidative stress; reactive oxygen species (ROS); transcription factor.

Figure 1.

Burkholderia thailandensis OstR. A, ostR-emrB genomic locus. Transposon insertion is indicated by inverted triangles. Lines below the arrows illustrating open reading frames represent the positions of PCR amplicons used for quantitative RT-PCR. B, predicted model of OstR based on template 4AIK, created by SwissModel (in automated mode; GMQE 0.55) and visualized by PyMOL. One monomer is shown in cyan and the other is in gray, with positively charged residues in blue and negatively charged amino acids in red. Serine at position 28 and alanine at position 164 (marking the range of amino acids included in the model) are shown in space-filling representation. Black arrow, DNA recognition helix. C, electrostatic surface potential was calculated using Swiss PDB Viewer (red, negative; blue, positive). D, magnified image of the positively charged patch. E, N- and C-terminal extensions of OstR that are not included in the modeled structure, with cysteines in boldface type and underlined.

Figure 2.

Regulation of gene expression. A, relative transcript level of emrB in WT, ΔostR, WTe (containing empty vector), and ΔostRc (complemented with ostR) strains. -Fold changes are reported relative to the reference gene encoding glutamate synthase large subunit (BTH_I3014/BTH_RS27550) and normalized to expression in WT cells, except for expression in ΔostRc, which is reported relative to WTe. Error bars, S.D. values of three biological replicates. B, relative -fold change in transcript level of ostR in ΔostR strain calculated by the 2^−ΔCT method relative to the reference gene and normalized to expression in WT cells. The gel shows expression of ostR in WT and ΔostR strains. Lane 1, 100-bp DNA marker. C and D, relative abundance of transcript level of the gene encoding EmrB in the presence of 2 mm H₂O₂, CuCl₂, and ZnCl₂ in WT and ΔostR strains. The transcript levels were calculated using 2^−ΔΔCT relative to the reference gene and normalized to expression in the corresponding unsupplemented cultures. Error bars, S.D. of three individual experiments. Asterisks represent statistically significant differences in expression compared with WT cells, except as indicated in A, based on a Student's t test (*, p < 0.05; **, p < 0.001).

Figure 3.

Gene deletions alter resistance to antibiotics and oxidants. In each panel, the top row shows WT, the middle row shows ΔostRX, and the bottom row shows ΔemrBX (strains deleted for antibiotic resistance cassettes within transposons). Spots represent 10-fold serial dilutions of the indicated strains; the doubling time of mutant strains (∼60 min) is greater than that of WT cells (∼26 min). A, LB. B, nalidixic acid (10 μg/ml). C, tetracycline (1 μg/ml). D, gentamicin (150 μg/ml). E, trimethoprim (50 μg/ml). F, treatment with 5 mm H₂O₂. G, 5 mm CuCl₂. H, 2 mm ZnCl₂.

Figure 4.

Expression of genes encoding AmrAB-OprA and BpeEF-OprC efflux pumps in the presence of tetracycline. A and B, expression of amrB (BTH_I2444) in ΔemrBX and ΔostRX, respectively. C and D, expression of bpeF (BTH_II2105) in ΔemrBX and ΔostRX, respectively. Expression is reported relative to unsupplemented cultures. Asterisks represent statistically significant differences in expression compared with unsupplemented cultures based on Student's t test (*, p < 0.05; **, p < 0.001). Horizontal axes identify the concentration of tetracycline (in μg/ml). Note that the ordinate scale for A differs from that in B–D. Error bars, S.D.

Figure 5.

Characterization of OstR. A, far-UV CD spectrum of OstR. Ellipticity is presented in machine units (millidegrees). B, thermal denaturation of OstR determined by differential scanning fluorometry. Fluorescence intensity reflects binding of SYPRO Orange to hydrophobic regions of denatured protein as a function of temperature. C, prediction of intrinsically disordered regions (blue) and likelihood of disordered regions participating in protein interactions (orange). The y axis reflects confidence score, with values >0.5 considered relevant. D, release of metal ion from denatured OstR determined by absorbance of PAR. Absorbance at 416 nm corresponds to uncomplexed PAR, whereas absorbance at 520 nm reflects formation of PAR–metal ion complex. E and F, OstR oxidation by H₂O₂ (E) and CuCl₂ (F). In both images, the left lanes show protein marker (kDa), lanes 2 show protein incubated with DTT (Rd; species migrating at ∼43 kDa is residual oxidized protein), and lanes 3 show air-oxidized protein (O₂). E, lanes 4–8, increasing concentration of H₂O₂ (10 μm to 2 mm). F, lanes 4–7, increasing concentration of CuCl₂ (10 μm to 2 mm).

Figure 6.

OstR binds emrB promoter DNA. A, EMSA showing reduced OstR binding emrB promoter DNA (0.2 nm). DNA was titrated with increasing concentration of OstR (1–200 nm; lanes 2–14). Lane 1, free DNA only. Free DNA and protein–DNA complex are shown as FD and C1–C3, respectively, at the right. B and E, EMSA showing oxidized OstR binding emrB promoter DNA. Reactions in lanes 1 contained free DNA only, and reactions in lanes 2 contained reduced OstR. Lanes 3–14, operator DNA (0.2 nm) titrated with increasing concentration (1–200 nm) of oxidized protein (oxidized with 2 mm H₂O₂ and 5 μm CuCl₂, respectively). Normalized complex fraction for reduced OstR (C) and normalized complex fraction for H₂O₂-oxidized OstR (D) are plotted as a function of OstR concentration. S.D. values of three replicates are represented as error bars. F, EMSA showing OstR (5 nm) bound to 0.2 nm labeled operator DNA challenged with increasing concentration of specific unlabeled 146-bp operator DNA (0.2–50 nm, lanes 3–8) or nonspecific DNA pET28b (0.2–10 nm, lanes 10–15). The reaction in lane 1 contained free DNA only; the reactions in lanes 2 and 9 contained no competitor DNA.

Figure 7.

Oxidation of OstR cysteine variants by H₂O₂ and CuCl₂. A, C, and E, OstR-C3A. OstR-C4A and OstR-C169A oxidized with increasing concentration of H₂O₂ (10 μm to 2 mm). B, D, and F, OstR-C3A. OstR-C4A and OstR-C169A oxidized with increasing concentration of CuCl₂ (10 μm to 2 mm). Lanes 1, protein marker (kDa); lanes 2, reduced protein (Rd); lanes 3, air-oxidized protein (O₂), except for B, in which air-oxidized protein is not shown.

Figure 8.

Cysteine variants bind DNA with reduced affinity. A, OstR-C3A binding emrB promoter DNA. DNA (0.2 nm) was titrated with increasing concentration of OstR-C3A (1–200 nm; lanes 2–13). Lane 1, free DNA only. Free DNA and protein–DNA complex are shown as FD and C1–C3, respectively, at the right. C and E, EMSA showing binding of OstR-C4 and OstR-C169A to promoter DNA, respectively. B, D, F, normalized complex fraction for OstR-C3A, OstR-C4A, and OstR-C169A, respectively, plotted as a function of protein concentration. Error bars, S.D. of three replicates.

Figure 9.

Differential scanning fluorometry showing thermal stability of cysteine variants. Fluorescence intensity reflects binding of SYPRO Orange to hydrophobic regions of denatured protein as a function of temperature. A, reduced OstR-C3A (red dotted line; left and bottom axes) and H₂O₂ oxidized OstR-C3A (blue dashed line; right and top axes). B, reduced OstR-C4A (teal dotted line; left and bottom axes) and H₂O₂-oxidized OstR-C4A (purple dashed line; right and top axes). C, reduced OstR-C169A (orange dotted line; left and bottom axes) and OstR-C169A bound to Zn²⁺ (green dashed line; right and top axes).

Figure 10.

All cysteine variants bind Zn²⁺. A–C, release of Zn²⁺ from denatured OstR-C3A, OstR-C4A, and OstR-C169A, respectively, determined by absorbance of Zn²⁺ in complex with PAR (peak absorbance at 520 nm). D, identified cysteine mutants were incubated with 2 mm ZnCl₂ for 30 min followed by treatment with 12 mm H₂O₂ for another 30 min (lanes 3, 5, and 7). Lanes 2, 4, and 6, Zn²⁺-treated OstR-C3A, OstR-C4A, and OstR-C169A, respectively.