Vitamin E Increases Antimicrobial Sensitivity by Inhibiting Bacterial Lipocalin Antibiotic Binding

Abstract

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes serious respiratory infections in patients with cystic fibrosis. Recently, we discovered that B. cenocepacia produces the extracellular bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures a range of antibiotics outside bacterial cells, providing a global extracellular mechanism of antimicrobial resistance. In this study, we investigated water-soluble and liposoluble forms of vitamin E as inhibitors of antibiotic binding by BcnA. Our results demonstrate that in vitro, both vitamin E forms bind strongly to BcnA and contribute to reduce the MICs of norfloxacin (a fluoroquinolone) and ceftazidime (a β-lactam), both of them used as model molecules representing two different chemical classes of antibiotics. Expression of BcnA was required for the adjuvant effect of vitamin E. These results were replicated in vivo using the Galleria mellonella larva infection model whereby vitamin E treatment, in combination with norfloxacin, significantly increased larva survival upon infection in a BcnA-dependent manner. Together, our data suggest that vitamin E can be used to increase killing by bactericidal antibiotics through interference with lipocalin binding.IMPORTANCE Bacteria exposed to stress mediated by sublethal antibiotic concentrations respond by adaptive mechanisms leading to an overall increase of antibiotic resistance. One of these mechanisms involves the release of bacterial proteins called lipocalins, which have the ability to sequester antibiotics in the extracellular space before they reach bacterial cells. We speculated that interfering with lipocalin-mediated antibiotic binding could enhance the efficacy of antibiotics to kill bacteria. In this work, we report that when combined with bactericidal antibiotics, vitamin E contributes to enhance bacterial killing both in vitro and in vivo. This adjuvant effect of vitamin E requires the presence of BcnA, a bacterial lipocalin produced by the cystic fibrosis pathogen Burkholderia cenocepacia Since most bacteria produce lipocalins like BcnA, we propose that our findings could be translated into making novel antibiotic adjuvants to potentiate bacterial killing by existing antibiotics.

Keywords: Gram-negative bacteria; antibiotic resistance; chronic infection; cystic fibrosis; intrinsic resistance; lipocalin; vitamin E.

FIG 1

bcoA, bcnA, and bcnB form an operon. (A) PCR amplification using cDNA and gDNA templates derived from B. cenocepacia K56-2. Lanes: M, 100-bp ladder; 1, gDNA-derived PCR product; 2, negative control (no RT during cDNA synthesis); 3, cDNA-derived PCR product. All samples were run in the same gel; the dotted white lines are for clarity only. (B) Intragenic amplicons are in black; intergenic regions are in red. The direction of transcription is indicated. Primers and PCR amplicon positions from panel A are indicated by the black (intragenic regions) and blue (intergenic regions) arrow pairs.

FIG 2

Vitamin E antibiotic adjuvant effect depends on the BcnA protein. (A) MIC of norfloxacin alone or in combination with 0.7 mM TPGS against Burkholderia strains, determined by broth microdilution in cation-adjusted Mueller-Hinton broth (MHB) at 24 h. Data points are from 4 independent experiments, each done in duplicate (n = 8). (B) MIC of ceftazidime alone or combined with TPGS against Burkholderia strains in MHB at 24 h (n = 4, from 2 independent experiments). (C) MIC of norfloxacin alone or mixed with 1.4 mM TPGS against ΔbcnA mutants carrying pDA17 and ΔbcnA mutants harboring pDA17 bcnA, determined by broth dilution methods in MHB at 24 h (n = 4, from 2 independent experiments). (D) MIC of ceftazidime alone or mixed with 1.4 mM TPGS against ΔbcnA mutants carrying pDA17 and ΔbcnA mutants harboring pDA17 bcnA at 24 h (n = 4, from 2 independent experiments). Results are shown as the mean MIC ± SEM, and P values were calculated by the paired t test.

FIG 3

Antibiotic challenge assays. Burkholderia strains were challenged with MIC₂₅ of norfloxacin, which varied for each strain (16 μg/ml for the parental strain K56-2 and the ΔbcnB strain, 8 μg/ml for ΔbcnAB and ΔbcoA strains, and 2 μg/ml for ΔbcnA and ΔbcnAB ΔbcoA strains) with or without 0.7 mM TPGS (A to F) in cation-adjusted MHB for 2 h at 37°C. For challenge with polymyxin B, bacteria were exposed to MIC₂₅ (256 μg/ml of polymyxin B for all strains), with or without 0.7 mM TPGS (G to L), using non-cation-adjusted MHB for 6 h at 37°C. (A and G) Wild-type K56-2; (B and H) ΔbcnA strain; (C and I) ΔbcnB strain; (D and J) ΔbcnAB double mutant; (E and K) ΔbcoA strain; (F and L) ΔbcnAB ΔbcoA triple mutant. Results are shown as the mean MIC ± SEM, by determining the CFU of surviving bacteria. P values were calculated by the paired t test. Data represent the results of 3 independent experiments, each done in duplicate (n = 6).

FIG 4

In vitro protection assays against norfloxacin with 1.5 μM BcnA protein in the absence or presence of 0.7 mM TPGS. The results were obtained from 4 independent experiments, each in duplicate (n = 8), and plotted as the mean MIC ± SEM. **, P = 0.02 determined by paired t test.

FIG 5

Binding analyses by microcalorimetry. (A and B) ITC data of TPGS binding to BcnA (A) and the presence of norfloxacin (B). The bottom panel shows that the binding isotherm is obtained by plotting the areas under the peaks in the top panel against the molar ratio of ligand (TPGS or norfloxacin) added to BcnA present in the cell. (C) Physicochemical binding parameters of TPGS and norfloxacin to BcnA. The binding affinity (K) and enthalpy change (ΔH°) were obtained from ITC profiles fitting to “one set of binding sites” modeled by Origin 7 software. ΔG° and TΔS° were determined by the equations ΔG° = − RT lnK and TΔS° = ΔH° − ΔG°. N is the binding stoichiometry. K_D is the dissociation constant. The data presented are the average from two experiments.

FIG 6

In vivo protection assays using G. mellonella infection model. (A) Larvae were infected with 8 × 10³ CFU of B. cenocepacia K56-2, ΔbcnA mutant, and ΔbcnA mutant plus 1.5 μM BcnA protein in the presence or absence of 0.7 mM TPGS. Larva survival was monitored over 72 h postinfection. (B) Hemolymph from at least three B. cenocepacia-infected larvae was extracted and pooled at 48 h postinfection to determine the bacterial CFU/ml by plating onto LB agar containing 200 μg/ml spectinomycin (to prevent the growth of the larvae’s endogenous microbial flora agar. The results represent the mean and the SEM from 3 independent biological replicates. ***, P < 0.001.

FIG 7

Treatment of G. mellonella-infected larvae with norfloxacin or norfloxacin plus TPGS in combination with the wild type or ΔbcnA mutant. Larvae were infected with 100 CFU of bacteria for 24 h and then treated with the MIC of norfloxacin appropriate to each bacterial strain with or without 1.7 mM TPGS. Percent survival and the bacterial load (CFU/ml) in the larva’s hemolymph were determined at 48 h postinfection. The results represent the mean and SEM for 3 biological repeats. (A and B) Percent survival and bacterial load, respectively, of larvae infected with the parental K56-2 strain. (C and D) Percent survival and bacterial load, respectively, of ΔbcnA strain-infected larvae.

FIG 8

Vitamin E can reduce the antibiotic resistance of P. aeruginosa PAO1. (A) MIC of norfloxacin and ceftazidime with and without 1.4 mM TPGS against PAO1 using microdilution method at 24 h (n = 4, from 2 independent experiments and 2 biological replicates). Results are shown as the mean MIC ± SEM and compared using the paired t test. (B) Norfloxacin-TPGS treatment of G. mellonella larvae infected with 10 CFU of P. aeruginosa PAO1 or B. cenocepacia K56-2 for 2 h and then treated with the MIC of norfloxacin, 1.7 mM TPGS, or the two together. Percentage of larva survival was monitored after 24 h. n = 3. *, P < 0.05; **, P < 0.01.