TY - JOUR
T1 - Mutations in fbiD (Rv2983) as a novel determinant of resistance to pretomanid and delamanid in mycobacterium tuberculosis
AU - Rifat, Dalin
AU - Li, Si Yang
AU - Ioerger, Thomas
AU - Shah, Keshav
AU - Lanoix, Jean Philippe
AU - Lee, Jin
AU - Bashiri, Ghader
AU - Sacchettini, James
AU - Nuermberger, Eric
N1 - Funding Information:
We gratefully acknowledge support in the form of funding from the Bill and Melinda Gates Foundation (OPP1037174) (E.N.) and the National Institutes of Health (R01-AI111992) (E.N.). G.B. is supported by a Sir Charles Hercus Fellowship through the Health Research Council of New Zealand.
Funding Information:
The Global Alliance for TB Drug Development kindly provided pretomanid and delamanid under a material transfer agreement. We gratefully acknowledge Anna Upton and Juliano Timm for critical reading of the manuscript. We gratefully acknowledge support in the form of funding from the Bill and Melinda Gates Foundation (OPP1037174) (E.N.) and the National Institutes of Health (R01-AI111992) (E.N.). G.B. is supported by a Sir Charles Hercus Fellowship through the Health Research Council of New Zealand. D.R. and E.N. conceived the study and designed the experiments. S.-Y.L. and J.-P.L. assisted with the design and conduct of the in vivo experiment. Whole-genome sequencing was performed and analyzed by T.I. and J.S., and the results were further analyzed by D.R. and E.N. In vitro experiments were performed and analyzed by D.R., K.S., J.L., and E.N. The manuscript was drafted by D.R. and E.N. with critical input from T.I., J.S., and G.B. E.N. reports research support in the form of a contract from the Global Alliance for TB Drug Development. E.N. and J.S. report research collaborations with the Global Alliance for TB Drug Development on U19-AI-142735. All other authors declare that they have no competing interests.
Publisher Copyright:
© 2020 American Society for Microbiology. All Rights Reserved.
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
KW - Cofactor F
KW - Mouse model
KW - Nitroimidazole
KW - Resistance
KW - Tuberculosis
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UR - http://www.scopus.com/inward/citedby.url?scp=85098657348&partnerID=8YFLogxK
U2 - 10.1128/AAC.01948-20
DO - 10.1128/AAC.01948-20
M3 - Article
C2 - 33077652
AN - SCOPUS:85098657348
SN - 0066-4804
VL - 65
JO - Antimicrobial agents and chemotherapy
JF - Antimicrobial agents and chemotherapy
IS - 1
M1 - e01948-20
ER -