Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

Abstract

The nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10^-5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F₄₂₀ synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F₄₂₀ and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F₄₂₀ biosynthesis. Finally, Rv2983 mutants and other F₄₂₀H₂-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Keywords: cofactor F420; mouse model; nitroimidazole; resistance; tuberculosis.

FIG 1

Selective amplification of spontaneous pretomanid-resistant mutants during pretomanid monotherapy in mice is dose dependent and is more pronounced in C3HeB/FeJ mice. After aerosol infection with M. tuberculosis H37Rv, BALB/c and C3HeB/FeJ mice were treated with a range of doses of pretomanid for 8 weeks and sacrificed at different time points before and after treatment for lung CFU counts. (A) Mean (± SD) total lung CFU counts on the day after infection (W-8), on the day of treatment initiation (D0), and after 3 weeks of treatment with the indicated pretomanid dose (in mg/kg of body weight). Dose-dependent bactericidal activity was observed in both strains. (B) Mean (± SD) total and pretomanid-resistant lung CFU counts in BALB/c mice on day 0 and after 8 weeks of treatment with the indicated pretomanid dose. Dose-dependent bactericidal activity and selection of pretomanid-resistant bacteria was observed, with the resistant population overtaking the susceptible population at doses ≥300 mg/kg. (C) Mean (± SD) total and pretomanid-resistant lung CFU counts in C3HeB/FeJ mice on day 0 and after 8 weeks of treatment with the indicated pretomanid dose. Dose-dependent bactericidal activity and selection of pretomanid-resistant bacteria were observed, with the resistant population overtaking the susceptible population at doses ≥30 mg/kg. *, P < 0.05; ***, P < 0.001.

FIG 2

Mutation frequencies and mutation types of genes associated with pretomanid resistance. WGS was performed with 136 pretomanid-resistant colonies and 25 colony pools picked from 47 individual mice harboring pretomanid-resistant CFUs after 8 weeks of treatment. A total of 99 unique mutations in these 6 genes were identified. (A) Overall mutation frequencies. (B) Mutation frequencies and mutation types in BALB/c mice. (C) Mutation frequencies and mutation types in C3HeB/FeJ mice.

FIG 3

Rv2983 is required for efficient F₄₂₀ synthesis from Fo. F₄₂₀ and Fo content measured in M. smegmatis strains harboring different recombinants relative to the control strain containing the empty vector pYUBDuet after 6 (A) and 26 (B) hours of 1 mM IPTG induction; F₄₂₀ (C) and Fo (D) content was measured in the Rv2983 mutant strains of M. tuberculosis and the control strains, including B101 (ΔRv2983, A198P), KA016 (ΔRv2983, Q114R), H37Rv (wild type), B101 complemented strain (pMH94-Rv2983), B101 complemented strain (pMH94-hsp60-Rv2983), KA026 (ΔfbiC, IS6110 insertion in 85 bp upstream of fbiC), and K91 (Δddn, IS6110 insertion in aa D108), after growth in 7H9 broth for 6 days. Schematic diagram (E) of proposed nitroimidazole activation pathway showing Rv2983 as FbiD catalyzing EPPG biosynthesis. Fo, 7,8-didemethyl-8-hydroxy-5-deazariboflavin; PEP, phosphoenolpyruvate; EPPG, enolpyruvyl-diphospho-5′-guanosine.

FIG 4

The F₄₂₀-deficient pretomanid-resistant Rv2983 mutant is hypersensitive to oxidative stress and progressive hypoxia but is not attenuated in BALB/c mouse lungs. (A) M. tuberculosis growth kinetics in 7H9 broth containing 20 μM menadione. (B) M. tuberculosis growth kinetics in 7H9 broth containing 100 μM menadione. (C) M. tuberculosis growth and survival under progressive hypoxia. (D) Lung CFU counts in BALB/c mice after aerosol infection with M. tuberculosis strains.

FIG 5

The F₄₂₀-deficient pretomanid-resistant Rv2983 mutant is hypersusceptible to anti-TB drugs. Time-kill kinetics was performed using M. tuberculosis strains in 7H9 broth containing the following drugs: INH of 0.15 μg/ml (A), linezolid (LZD) of 2.5 μg/ml (B), bedaquiline (BDQ) of 0.3 μg/ml (C), and cefazolin (CFZ) of 1.84 μg/ml (D). The difference in CFU/ml was calculated based on the CFU/ml at each time point relative to that on day 0 (after subculture of the strains to a drug-containing medium).

FIG 6

F₄₂₀H₂-deficient pretomanid-resistant mutants of M. tuberculosis are more susceptible to growth inhibition by malachite green. (A) Growth of wild-type M. tuberculosis on 7H9 agar is inhibited by malachite green (MG) in a concentration-dependent manner. F₄₂₀H_2-deficient, pretomanid-resistant M. tuberculosis mutants (fbiA and C, fgd, and Rv2983) are inhibited at lower MG concentrations than those of the wild type and the F₄₂₀H₂-sufficient, pretomanid-resistant ddn mutant. (B) Complementation of the B101 mutant with wild-type Rv2983 restores tolerance to MG after 28 days of incubation.

FIG 7

A mutation in Rv2983 causes growth inhibition on commercial 7H10 agar and LJ slants but not on commercial 7H11 agar. Aliquots of M. tuberculosis cultures were spread onto various solid media purchased commercially after serial 10-fold dilutions. (A and B) Mean CFU counts on 7H10 (A) and 7H11 (B) agar plates after 21, 28, and 35 days of incubation. (C) Colonies on LJ slants inoculated with serially diluted aliquots after 28 and 35 days of incubation. 1, H37Rv wild type; 2, B101 mutant (ΔRv2983, A198P); 3, B101 mutant complemented with Rv2983 behind the native promoter; 4, B101 mutant complemented with Rv2983 behind the hsp60 promoter; 5, K91 mutant (Δddn, IS6110 insertion in D108).