Mutations in pepQ Confer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

Abstract

The novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations in Rv0678 that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance in Mycobacterium tuberculosis: loss-of-function mutations in pepQ (Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase. pepQ mutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression of Rv0678, mmpS5, or mmpL5 between mutant and parent strains. Complementation of a pepQ mutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility both in vitro and in mice. Although the mechanism by which mutations in pepQ confer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

FIG 1

Mean lung log₁₀ CFU counts (±SD) after 4 and 8 weeks of treatment with bedaquiline- and clofazimine-containing regimens. The main graph does not include results from mice in which bedaquiline and clofazimine cross-resistant mutants were selected after 8 weeks of treatment: 2 and 3 mice from the B and BC groups with mean lung CFU counts of 3.22 ± 0.16 and 2.12 ± 0.69 log₁₀, respectively (inset). The CFU counts at the time of treatment initiation are indicated at day 0. Abbreviations: R, rifampin; H, isoniazid; Z, pyrazinamide; B, bedaquiline; C, clofazimine; L, linezolid; Pa, pretomanid; E, ethambutol; M, moxifloxacin.

FIG 2

Mean lung log₁₀ CFU counts (±SD) (A and B) and change in CFU counts (week 4 − day 0) (C and D) after 4 weeks of treatment in mice infected with the parental H37Rv strain (A and C) or the B5 mutant (B and D). CFU counts were not determined for untreated mice infected with the parental strain, which required euthanasia prior to the predetermined endpoint. Abbreviations: UT, untreated; H, isoniazid (10 mg/kg); C, clofazimine (20 mg/kg); B, bedaquiline (12.5, 25, or 50 mg/kg).

FIG 3

Mean lung log₁₀ CFU counts (±SD) (A to C) and change in lung CFU counts (week 4 − day 0) (D to F) after 4 weeks of treatment in mice infected with the parental H37Rv strain (A and D), the B5 mutant (B and E), or the complemented B5::pepQ strain (C and F). Abbreviations: UT, untreated; H, isoniazid (10 mg/kg); C, clofazimine (20 mg/kg); B, bedaquiline (25 mg/kg).

FIG 4

Locations of mutations and domains in pepQ.

FIG 5

Multiple amino acid sequence alignment for the products of M. tuberculosis pepQ, Lactococcus lactis pepP, and E. coli pepP, constructed using ClustalW. Residues coordinating the Mn²⁺ ions are shown in red. Asterisks indicate positions which have a single, fully conserved residue. Colons indicate conservation between groups of strongly similar properties, scoring >0.5 in the Gonnet PAM 250 matrix. Periods indicate conservation between groups of weakly similar properties, scoring ≤0.5 in the Gonnet PAM 250 matrix.