Acquired Resistance of <i>Mycobacterium tuberculosis</i> to Bedaquiline

<div><p>Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. <i>In vitro</i> resistance to BDQ was previously shown to be due to target-based mutations. Here we report that non-target based resistance to BDQ, and cross-resistance to clofazimine (CFZ), is due to mutations in Rv0678, a transcriptional repressor of the genes encoding the MmpS5-MmpL5 efflux pump. Efflux-based resistance was identified in paired isolates from patients treated with BDQ, as well as in mice, in which it was confirmed to decrease bactericidal efficacy. The efflux inhibitors verapamil and reserpine decreased the minimum inhibitory concentrations of BDQ and CFZ <i>in vitro</i>, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance <i>in vivo</i>. Cross-resistance between BDQ and CFZ may have important clinical implications.</p></div

Translational effect of non-target based resistance to BDQ in mice and inability of verapamil to reverse it.

<p>Log kill values of wild type H37Rv and <i>Rv0678</i> mutants in mice treated with BDQ at 6.25 or 50 mg/kg (5x/week), in the presence (25 V) or absence (0 V) of 25 mg/kg verapamil for 4 weeks. Values represent the median log reduction versus baseline CFU for 6 mice.</p

Top 10 upregulated proteins of the BDQ-resistant strains EH3.6 and EH 3.2 compared to EH3.0.

<p>Proteins displaying the highest differential upregulation factors (top 10 proteins) are indicated. The descriptions of the genes were retrieved from TubercuList (<a href="http://tuberculist.epfl.ch/" target="_blank">http://tuberculist.epfl.ch/</a>).</p

Mechanism of BDQ and CFZ resistance in <i>Rv0678</i> mutants.

<p><b>A.</b> Regulation of <i>mmpS5</i> and <i>mmpL5</i> transcription by wild type Rv0678 repressor. Rv0678 protein binds to the intergenic region located between <i>Rv0678</i> and <i>mmpS5</i>, which contains the-10 consensus boxes of promoters for both <i>Rv0678</i> and <i>mmpS5</i>. This prevents the RNA polymerase to start transcription, resulting in the decrease of expression of MmpS5, MmpL5 and Rv0678 proteins. In response to an unknown stimulus, the regulator detaches from DNA and transcription can be resumed. <b>B.</b> Lack of regulation in <i>Rv0678</i> mutants. The strains carrying frame shifting mutations in <i>Rv0678</i> will not produce a functional repressor, thus the transcription of <i>mmpS5, mmpL5</i> and <i>Rv0678</i> will be increased. If the mutation results in an amino acid polymorphism, the protein may still be functional, but with reduced DNA-binding ability depending on the location of the mutation. In either case, the final consequence will be an increase in the expression levels of the proteins MmpS5, MmpL5 and Rv0678.</p

MICs of MmpS5 and MmpL5 overexpression strains.

<p>MICs of strains overexpressing MmpS5, MmpL5 or the operon MmpS5-MmpL5 in the absence and presence of verapamil (40 µg/ml) are indicated. Each value is the average of at least 2 independent experiments. <b>BDQ</b>: bedaquiline; <b>CFZ</b>: clofazimine; <b>VER</b>: verapamil.</p

Regulation of efflux pumps proteins in EH3.6 and EH3.2.

<p>Efflux pump proteins were selected from the complete dataset of the proteome analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102135#pone.0102135.s003" target="_blank">Table S1</a>). The descriptions of the genes were retrieved from TubercuList (<a href="http://tuberculist.epfl.ch/" target="_blank">http://tuberculist.epfl.ch/</a>).</p

Mutations in <i>Rv0678</i> gene and MICs of BDQ of resistant preclinical strains.

<p>The MICs of BDQ-R preclinical strains derived from either the drug susceptible H37Rv or the MDR EH3.0 parent strains are shown. Fold-changes between brackets represent the difference between resistant and wild-type MICs. <b>wt</b>: wild-type; <b>Ins</b>: insertion; <b>nt</b>: nucleotide<b>; BDQ</b>: bedaquiline; <b>BDQ-R</b>: bedaquiline-resistant.</p

Mutations in <i>Rv0678</i> gene of <i>M. tuberculosis</i> BDQ resistant strains.

<p><b>A.</b> PCR fragment amplified for sequencing and mapping of mutations in <i>Rv0678</i> gene of BDQ resistant strains in (<b>B</b>) H37Rv-derived mutants and (<b>C</b>) EH 3.0-derived mutants. Codons START and STOP of <i>Rv0678</i> gene are underlined. The nucleotide positions are indicated on top of each mutation. Mutations are bold and colored: missense mutations are indicated in green, insertions are highlighted in red, and the 1.3 Kb insertion sequence IS<i>6110</i> is colored in blue. The direct repeats of IS<i>6110</i> are indicated in italics.</p

MICs of BDQ and CFZ of preclinical strains with EPIs.

<p>MICs of BDQ and CFZ of preclinical strains, in the absence and presence of verapamil (40 µg/ml) or reserpine (3 µg/ml). Each value is the average of 3 independent experiments. For MIC without EPIs, fold-changes between brackets represent the difference between resistant and wild-type MICs. For MICs in the presence of EPIs, fold-changes between brackets represent the differences between MIC with and without EPI. <b>BDQ</b>: bedaquiline; <b>CFZ</b>: clofazimine; <b>RES</b>: reserpine; <b>VER</b>: verapamil.</p