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bioRxiv · 10.1101/2020.09.10.292508

Mutations in fbiD(Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis

Rifat, D.·Li, S.-Y.·Ioerger, T. R.·Shah, K.·Lanoix, J.-P.·Lee, J.·Bashiri, G.·Sacchettini, J. C.·Nuermberger, E.
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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, 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.

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Rifat, D., Li, S.-Y., Ioerger, T. R., Shah, K., Lanoix, J.-P., Lee, J., Bashiri, G., Sacchettini, J. C., Nuermberger, E.. 2020-09-11. Mutations in fbiD(Rv2983) as a novel determinant of resistance to pretomanid and delamanid in Mycobacterium tuberculosis. https://doi.org/10.1101/2020.09.10.292508

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