Understanding Carbapenem Translocation through OccD3 (OpdP) of <i>Pseudomonas aeruginosa</i>

<i>Pseudomonas aeruginosa</i> utilizes a plethora of substrate specific channels for the uptake of small nutrients. OccD3 (OpdP or PA4501) is an OprD-like arginine uptake channel of <i>P. aeruginosa</i> whose role has been implicated in carbapenem uptake. To understand the mechanism of selective permeation, we reconstituted single OccD3 channels in a planar lipid bilayer and characterized the interaction with Imipenem and Meropenem, analyzing the ion current fluctuation in the presence of substrates. We performed point mutations in the constriction region of OccD3 to understand the binding and translocation of antibiotic in OccD3. By mutating two key residues in the substrate binding sites of OccD3 (located in the internal loop L7 and basic ladder), we emphasize the importance of these residues. We show that carbapenem antibiotics follow a similar path as arginine through the constriction zone and the basic ladder to translocate across OccD3

Purification and immunodetection of the <i>Bps</i>Omp38 variants, expressed in <i>E. coli</i>.

<p>(A) Chromatographic profile of <i>Bps</i>Omp38 purification with a Hitrap SP HP column, connected to an ÄKTA Prime plus FPLC system. The protein was eluted with a linear gradient of 0–1 M KCl, (B) SDS/PAGE followed by Coomassie Blue staining, (C) immunoblot analysis of the same samples as in panel B, using polyclonal anti-<i>Bps</i>Omp38 serum, (D) immunoblot analysis using anti-His₆ monoclonal antibody, and (E) identification of tryptic digests of the expressed proteins by nanoLC/ESI MS (First BASE Laboratories, Malaysia). Eight peptides, designated P1–P8 and shown in red, were unambiguously identified by MASCOT as identical to internal sequences of <i>Bps</i>Omp38. The 20-aminoacid signal peptide is underlined. Lanes: Std, standard proteins; 1, <i>Bps</i>Omp38WT; 2, <i>Bps</i>Omp38Y119A; 3, <i>Bps</i>Omp38 mY119F; 4, refolded <i>Bps</i>Omp38; 5, <i>E. coli</i> OmpF; 6, <i>E. coli</i> OmpN; 7, and <i>V. harveyi</i> ChiP.</p

Ion current recordings obtained by the black lipid membrane (BLM) reconstitution technique.

<p>BLM measurements of successive insertions of <i>Bps</i>Omp38 trimers at a transmembrane potential of +100 mV. Lipid bilayers were formed across a 200-µM aperture by the ‘painting’ technique using 50 mg.mL⁻¹ azolectin in <i>n</i>-hexane and bathed on either side in 1 M KCl. <i>Bps</i>Omp38 (1 µg.mL⁻¹) was added on the <i>cis</i> side. (A) <i>Bps</i>Omp38WT, (B) <i>Bps</i>Omp38Y119A, and (C) <i>Bps</i>Omp38Y119F. Left panels are ion current traces acquired for 100 s. Fast insertion of one <i>Bps</i>Omp38WT channel, occurring within the millisecond time-resolution, was captured and shown as an inset. The traces represent multiple insertions of <i>Bps</i>Omp38 variants produced by applied membrane potentials of +100 mV. Right panels are histograms from the corresponding traces, giving the probability of a pore conductance (G) averaged over several hundred inserting channels as indicated. The black line represents a single Gaussian fit.</p

Summary of relative permeabilities of <i>Bps</i>Omp38 and the levels of <i>Bps</i> resistance to cephalosporin and carbapenem, together with the structures of the drugs.

<p>The value in brackets is charge state of each antibiotic at pH<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone.0095918-Irwin1" target="_blank">[35]</a>.</p

Swelling of <i>Bps</i>Omp38 proteoliposomes induced by cephalosporins and carbapenems.

<p>The proteoliposomes were prepared at pH–6 independent experiments. Levels of <i>Bps</i> resistance were determined by a broth micro dilution assay as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone-0095918-t001" target="_blank">Table 1</a>. R represents resistant; S, sensitive; and I, intermediate. Differences in the three data sets were evaluated using one-way ANOVA. Statistically significant differences (P<0.05) are marked with an asterisk (*). Values are as means ± SD, obtained from 4–6 independent sets of experiments.</p

A ribbon representation of the homology model of <i>Bps</i>Omp38.

<p>The modeled structure of <i>Bps</i>Omp38WT (A) shows the key residue Y119 (red) protruding into the channel lumen (top view). This residue was mutated to Ala or Phe, generating two single mutants, <i>Bps</i>Omp38Y119A (B) and <i>Bps</i>Omp38Y119F (C).</p

Antibiotic susceptibility of a clinically derived strain of <i>Burkholderia pseudomallei</i>.

<p>The values presented are obtained from the experiments performed 4–6 times.</p>a<p>Breakpoints defined for <i>Pseudomonas spp</i>. follow the EUCAST Clinical Breakpoint Table v. 4.0, valid from 2014-01-01 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone.0095918-Clinical1" target="_blank">[26]</a>. R, Resistant; S, Sensitive; NA, Not applicable;</p><p>-, No breakpoints. Susceptibility testing is not recommended. In order to simplify the EUCAST tables, the intermediate category is not listed.</p

Ion current recordings obtained by the black lipid membrane (BLM) reconstitution technique.

<p>BLM measurements of successive insertions of <i>Bps</i>Omp38 trimers at a transmembrane potential of +100 mV. Lipid bilayers were formed across a 200-µM aperture by the ‘painting’ technique using 50 mg.mL⁻¹ azolectin in <i>n</i>-hexane and bathed on either side in 1 M KCl. <i>Bps</i>Omp38 (1 µg.mL⁻¹) was added on the <i>cis</i> side. (A) <i>Bps</i>Omp38WT, (B) <i>Bps</i>Omp38Y119A, and (C) <i>Bps</i>Omp38Y119F. Left panels are ion current traces acquired for 100 s. Fast insertion of one <i>Bps</i>Omp38WT channel, occurring within the millisecond time-resolution, was captured and shown as an inset. The traces represent multiple insertions of <i>Bps</i>Omp38 variants produced by applied membrane potentials of +100 mV. Right panels are histograms from the corresponding traces, giving the probability of a pore conductance (G) averaged over several hundred inserting channels as indicated. The black line represents a single Gaussian fit.</p

Single channel recordings of <i>Bps</i>Omp38 porin in artificial lipid membranes.

<p>Typical ion current traces of single <i>Bps</i>Omp38 variant channels in a fully open state at a transmembrane potential of +100 mM, (B) Current-voltage relationship of <i>Bps</i>Omp38WT in comparison with its two mutants. The current through a single open <i>Bps</i>Omp38 channel was monitored in 1 M KCl, following discrete changes in the voltage across the phospholipid membrane, from −100 to +125 mV. The slopes of a linear fit yielded the single channel conductances of individual <i>Bps</i>Omp38 channels. Data points for <i>Bps</i>Omp38WT, <i>Bps</i>OmpY119A and <i>Bps</i>OmpY119F are plotted as circles, squares and triangles, respectively. Differences in the three data sets were evaluated using one-way ANOVA. Statistically significant difference (P<0.05) is shown with an asterisk (*).</p

Statistical analysis of MIC values of <i>Bps</i> shown in Table 1 by one-way ANOVA and log₂ dilution methods.

a<p>N is the total number of samples used for both analyses. Two equal-sized sampling groups (each group, n = 6) were used, in the absence and presence of PAβN.</p>b<p>NS represents non-significant difference between the two studied groups at <i>p</i><0.05.</p>c<p>NS represents non-significant difference between the two studied groups at essential agreement ≥ 85% <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095918#pone.0095918-Marley1" target="_blank">[37]</a>.</p