Predicting nitroimidazole antibiotic resistance mutations in Mycobacterium tuberculosis with protein engineering

Our inability to predict which mutations could result in antibiotic resistance has made it difficult to rapidly identify the emergence of resistance, identify pre-existing resistant populations, and manage our use of antibiotics to effectively treat patients and prevent or slow the spread of resistance. Here we investigated the potential for resistance against the new antitubercular nitroimidazole prodrugs pretomanid and delamanid to emerge in Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Deazaflavin-dependent nitroreductase (Ddn) is the only identified enzyme within M. tuberculosis that activates these prodrugs, via an F420H2-dependent reaction. We show that the native menaquinone-reductase activity of Ddn is essential for emergence from hypoxia, which suggests that for resistance to spread and pose a threat to human health, the native activity of Ddn must be at least partially retained. We tested 75 unique mutations, including all known sequence polymorphisms identified among similar to 15,000 sequenced M. tuberculosis genomes. Several mutations abolished pretomanid and delamanid activation in vitro, without causing complete loss of the native activity. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, before being exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with structural analysis that indicates delamanid and pretomanid bind to Ddn differently. We suggest that the mutations identified in this work be monitored for informed use of delamanid and pretomanid treatment and to slow the emergence of resistance. Bacterial pathogens often evolve resistance to antibiotics via mutations in the coding sequences of genes-frequently the target, an enzyme that metabolizes or transports the active drug, or an enzyme that activates a prodrug. In the case of tuberculosis, antibiotic resistance is a growing problem, with the rapid emergence of multi-drug resistant strains. New nitroimidazole-based antibiotic prodrugs, such as pretomanid and delamanid have the potential to help infected individuals, but we must guard against the evolution of resistance to these new compounds in Mycobacterium tuberculosis. In this report we use protein engineering to identify mutations that could potentially result in antibiotic resistance by knocking out the prodrug activating activity of the deazaflavin dependent nitroreductase (DDN), without completely abolishing its native menaquinone reductase activity. The retention of its native activity is important as DDN appears to be required for M. tuberculosis to emerge from hypoxia. Strikingly, when we analysed similar to 15,000 M. tuberculosis genomes from clinical strains, we identified several that harboured mutations that we identified as abolishing prodrug activation in vitro. A hypervirulent Beijing strain N0008 from Vietnam (which had not been exposed to pretomanid in the clinic) was predicted, and confirmed, to be resistant to pretomanid, revealing that resistance to this drug has arisen through genetic drift and not selective pressure in this instance. These data show that by testing potential resistant mutations in the laboratory before large-scale use of antibiotics, we should be able to use them more judiciously in order to slow the spread of resistance.