Mutations in panD encoding aspartate decarboxylase are associated with pyrazinamide resistance in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug susceptible and multidrug-resistant tuberculosis (MDR-TB). Resistance to PZA is most commonly associated with mutations in the pncA gene encoding nicotinamidase/pyrazinamidase which converts the prodrug PZA to the active form pyrazinoic acid (POA). RpsA (ribosomal protein S1) involved in trans-translation was recently shown to be a target of PZA and mutations in RpsA are found in some PZA-resistant TB strains. However, some other PZA-resistant strains lack mutations in either pncA or rpsA. To identify potential new mechanisms of PZA resistance, we isolated 174 in vitro mutants of M. tuberculosis H37Rv resistant to PZA to search for resistant isolates that do not have pncA or rpsA mutations. DNA sequencing revealed that 169 of the 174 (97.1%) PZA-resistant mutants had pncA mutations but 5 mutants lacked pncA or rpsA mutations. Whole genome sequencing analyses revealed that the 5 PZA-resistant mutants had different mutations all occurring in the same gene panD encoding aspartate decarboxylase, which is involved in synthesis of beta-alanine that is a precursor for pantothenate and co-enzyme A biosynthesis. panD mutations were identified in naturally PZA-resistant Mycobacterium canetti strain and a PZA-resistant MDR-TB clinical isolate. Future studies are needed to address the role of panD mutations in PZA resistance and confirm PanD as a new target of PZA.