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

Abstract

The nitroimidazole pro-drugs 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, 91% of which occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance: 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.

Genetic diversity of candidate loci linked to Mycobacterium tuberculosis resistance to bedaquiline, delamanid and pretomanid

“…Mutations in these 3 genes have been shown to alter the production of F 420 22 . Similarly, it has been recently demonstrated the essential role of FbiD for the biosynthesis of F 420 and thereby its participation in DLM and PTM resistance 23 . The contribution of ndh , a NADH dehydrogenase, in isoniazid and ethionamide resistance involves retaining an appropriate NADH/NAD + ratio that enables the formation of adducts with NAD + , necessary for their activity 25 .…”

“…As pro-drugs, the nitroimidazoles DLM and PTM require activation by the deazaflavin (F 420 )-dependent nitroreductase Ddn. Mutations in the essential genes required for the F 420 cofactor biosynthesis and recycling, including ddn , fgd1 , fbiA , fbiB , fbiC , and fbiD , are putative resistance markers that directly hamper DLM/PTM activation or, work indirectly through F 420 depletion 5 , 21 – 23 . Important residues for the interaction of Ddn-PTM are known, which may differ from those involved in Ddn-DLM activation 24 .…”

Phylogenomic analysis and Mycobacterium tuberculosis antibiotic resistance prediction by whole-genome sequencing from clinical isolates of Caldas, Colombia

“…fbiC encodes a protein that participates in F 420 biosynthesis and harbors key mutations found in resistant MTB isolates. The mutation Ala132Val we detected in one isolate was not described in the systematic review on DMN resistance by Kadura et al ., [ 43 ] however, it showed a deleterious effect on the protein according to the PROVEAN score (-3,392); thus, the presence of this mutation in TB isolates not previously exposed to the drug requires future surveillance as not seems to be lineage-restricted [ 44 ].…”

The pipeline of new molecules and regimens against drug-resistant tuberculosis

“…In vitro frequency of spontaneous resistance development is high and comparable to isoniazid, as the enzymes involved in activation of the prodrug are not essential [49] , [50] . Resistance mutations are associated with genes involved the bioreductive activation of the drug within MTB (ddn ( Rv3547 ), fgd1) or the cofactor F420 biosynthesis (fbiA, fbiB, fbiC, fbiD) and most confer cross-resistance to pretomanid [50] .…”

“…This experience supported the design of pretomanid-based regimens of shortened treatment duration in Nix-TB and SimpliciTB [35] , [56] , [59] , [60] . Resistance mutations are associated with genes involved in the bioreductive activation of the drug as described for delamanid [50] .…”