Acquired resistance of Mycobacterium tuberculosis to bedaquiline

Abstract

Bedaquiline (BDQ), an ATP synthase inhibitor, is the first drug to be approved for treatment of multi-drug resistant tuberculosis in decades. In vitro 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 in vitro, but verapamil failed to increase the bactericidal effect of BDQ in mice and was unable to reverse efflux-based resistance in vivo. Cross-resistance between BDQ and CFZ may have important clinical implications.

Figure 1. Mutations in Rv0678 gene of M. tuberculosis BDQ resistant strains.

A. PCR fragment amplified for sequencing and mapping of mutations in Rv0678 gene of BDQ resistant strains in (B) H37Rv-derived mutants and (C) EH 3.0-derived mutants. Codons START and STOP of Rv0678 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 IS6110 is colored in blue. The direct repeats of IS6110 are indicated in italics.

Figure 2. Translational effect of non-target based resistance to BDQ in mice and inability of verapamil to reverse it.

Log kill values of wild type H37Rv and Rv0678 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.

Figure 3. Mechanism of BDQ and CFZ resistance in Rv0678 mutants.

A. Regulation of mmpS5 and mmpL5 transcription by wild type Rv0678 repressor. Rv0678 protein binds to the intergenic region located between Rv0678 and mmpS5, which contains the-10 consensus boxes of promoters for both Rv0678 and mmpS5. 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. Lack of regulation in Rv0678 mutants. The strains carrying frame shifting mutations in Rv0678 will not produce a functional repressor, thus the transcription of mmpS5, mmpL5 and Rv0678 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.