Gene Sequencing for Routine Verification of Pyrazinamide Resistance in Mycobacterium tuberculosis: a Role for pncA but Not rpsA

Public Health Ontario, Public Health Laboratories, Toronto, Ontario, Canada.
Journal of clinical microbiology (Impact Factor: 3.99). 08/2012; 50(11):3726-8. DOI: 10.1128/JCM.00620-12
Source: PubMed


Pyrazinamide (PZA) is an important component of first-line therapy for the treatment of tuberculosis. Here, we evaluate targeted gene sequencing as a supplement to phenotypic PZA susceptibility testing of Mycobacterium tuberculosis. Routine sequencing of pncA, but not rpsA, is effective for verification of PZA susceptibility results.

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    • "Recently, we identified ribosomal protein S1 (RpsA, Rv1630), a vital protein involved in trans-translation, as a target of PZA.7 Mutations in rpsA have been found in some PZA-resistant clinical isolates lacking pncA mutations.7,8,9,10 However, some PZA-resistant strains do not have mutations in either the pncA or rpsA genes,3,9,11 indicating the presence of a possible new resistance mechanism or target of PZA. "
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    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.
    Emerging Microbes and Infections 08/2014; 3(8):e58. DOI:10.1038/emi.2014.61 · 2.26 Impact Factor
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    • "Mutations in rpsA have been found in some PZA-resistant strains without pncA mutations.14,15,16 However, some PZA-resistant strains, which are typically low level PZA resistant (MIC=200–300 µg/mL, PH 6.0) and PZase positive do not have mutations in either pncA or the rpsA gene.12,15,17 To identify new mechanisms of PZA resistance, in this study, we isolated a large number of in vitro generated mutants resistant to PZA and characterized these strains for novel mutations in their genomes by whole genome sequencing. "
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    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). 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 β-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.
    Emerging Microbes and Infections 06/2013; 2(6). DOI:10.1038/emi.2013.38 · 2.26 Impact Factor

  • Journal of clinical microbiology 01/2013; 51(1):383. DOI:10.1128/JCM.02760-12 · 3.99 Impact Factor
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