title: Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.
abstract: 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). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21205, USA.
authors.forename: Wanliang
authors.initials: W
authors.lastname: Shi
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University , Shanghai 200040, China.
authors.forename: Jiazhen
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21205, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Feng
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University , Shanghai 200040, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Cui
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21205, USA.
authors.forename: Shuo
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University , Shanghai 200040, China.
authors.forename: Xinhua
authors.initials: X
authors.lastname: Weng
authors.affiliation: Department of Infectious Diseases, Huashan Hospital, Fudan University , Shanghai 200040, China.
authors.forename: Wenhong
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21205, USA ; Department of Infectious Diseases, Huashan Hospital, Fudan University , Shanghai 200040, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
keywords: Pyrazinamide; aspartate decarboxylase; mode of action; panD; pyrazinoic acid resistance