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Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

Shi W, Chen J, Feng J, Cui P, Zhang S, Weng X, Zhang W, Zhang Y - Emerg Microbes Infect (2014)

Bottom Line: 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.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%).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21205, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of β-alanine concentration on POA susceptibility. M. tuberculosis H37Ra on 7H11 agar with no β-alanine (A), 0.1 µM β-alanine (B), 1 µM β-alanine (C), 5 µM β-alanine (D), 10 µM β-alanine (E), and 100 µM β-alanine (F).
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fig4: Effect of β-alanine concentration on POA susceptibility. M. tuberculosis H37Ra on 7H11 agar with no β-alanine (A), 0.1 µM β-alanine (B), 1 µM β-alanine (C), 5 µM β-alanine (D), 10 µM β-alanine (E), and 100 µM β-alanine (F).

Mentions: To identify the minimum concentration of β-alanine or pantothenate that antagonizes POA activity, M. tuberculosis strain H37Ra was plated on 7H11 plates containing various concentrations of β-alanine or pantothenate (0, 0.1, 1, 5, 10 and 100 µM). The results showed that the parent strain H37Ra was susceptible to 200 µg/mL POA (Figure 4A) and 0.1 µM β-alanine did not appear to antagonize POA activity (Figure 4B). The minimum concentration of β-alanine that showed obvious POA antagonism was 1 µM (Figure 4C), and the antagonism increased with the concentration of β-alanine (Figures 4C–4F). Interestingly, there was not much variation in the level of β-alanine antagonism across the range of 1–100 µM as all concentrations caused resistance to 600–800 µg/mL POA (Figures 4C–4F). Similarly, we evaluated the effect of pantothenate on POA susceptibility and found that concentration required to cause resistance to POA was an order of magnitude higher for pantothenate (10 µM) than β-alanine (1 µM) (Figure 5).


Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis.

Shi W, Chen J, Feng J, Cui P, Zhang S, Weng X, Zhang W, Zhang Y - Emerg Microbes Infect (2014)

Effect of β-alanine concentration on POA susceptibility. M. tuberculosis H37Ra on 7H11 agar with no β-alanine (A), 0.1 µM β-alanine (B), 1 µM β-alanine (C), 5 µM β-alanine (D), 10 µM β-alanine (E), and 100 µM β-alanine (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150287&req=5

fig4: Effect of β-alanine concentration on POA susceptibility. M. tuberculosis H37Ra on 7H11 agar with no β-alanine (A), 0.1 µM β-alanine (B), 1 µM β-alanine (C), 5 µM β-alanine (D), 10 µM β-alanine (E), and 100 µM β-alanine (F).
Mentions: To identify the minimum concentration of β-alanine or pantothenate that antagonizes POA activity, M. tuberculosis strain H37Ra was plated on 7H11 plates containing various concentrations of β-alanine or pantothenate (0, 0.1, 1, 5, 10 and 100 µM). The results showed that the parent strain H37Ra was susceptible to 200 µg/mL POA (Figure 4A) and 0.1 µM β-alanine did not appear to antagonize POA activity (Figure 4B). The minimum concentration of β-alanine that showed obvious POA antagonism was 1 µM (Figure 4C), and the antagonism increased with the concentration of β-alanine (Figures 4C–4F). Interestingly, there was not much variation in the level of β-alanine antagonism across the range of 1–100 µM as all concentrations caused resistance to 600–800 µg/mL POA (Figures 4C–4F). Similarly, we evaluated the effect of pantothenate on POA susceptibility and found that concentration required to cause resistance to POA was an order of magnitude higher for pantothenate (10 µM) than β-alanine (1 µM) (Figure 5).

Bottom Line: 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.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%).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University , Baltimore, MD 21205, USA.

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.

No MeSH data available.


Related in: MedlinePlus