Limits...
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

Antagonism of POA's activity against M. tuberculosis by pantothenate and β-alanine. (A) M. tuberculosis parent strain H37Ra on 7H11 agar, (B) a POA-resistant mutant with a PanD mutation (M117I), (C) parent strain on 7H11 agar containing 0.1 mM calcium pantothenate, (D) parent strain with 0.1 mM β-alanine, (E) parent strain with 0.1 mM L-alanine, (F) parent strain with 5 mM calcium L-aspartate, (G) parent strain with 10 mM L-valine, (H) parent strain with 10 mM glutamate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150287&req=5

fig3: Antagonism of POA's activity against M. tuberculosis by pantothenate and β-alanine. (A) M. tuberculosis parent strain H37Ra on 7H11 agar, (B) a POA-resistant mutant with a PanD mutation (M117I), (C) parent strain on 7H11 agar containing 0.1 mM calcium pantothenate, (D) parent strain with 0.1 mM β-alanine, (E) parent strain with 0.1 mM L-alanine, (F) parent strain with 5 mM calcium L-aspartate, (G) parent strain with 10 mM L-valine, (H) parent strain with 10 mM glutamate.

Mentions: Because PanD (aspartate decarboxylase) is involved in converting L-aspartic acid to β-alanine, a precursor for pantothenate and coenzyme A biosynthesis, we wondered if supplementation with β-alanine or pantothenate might render M. tuberculosis resistant to POA. To test this, pantothenate, β-alanine, and different amino acids that served as controls, were incorporated into 7H11 agar containing different concentrations of POA (200–800 µg/mL pH 6.8) followed by inoculation with M. tuberculosis H37Ra. The results showed that the parent strain and the mutant strain were sensitive and resistant to POA, respectively, on 7H11 plates containing different concentrations of POA (Figures 3A and 3B), but susceptibility to POA dramatically decreased when pantothenate or β-alanine (Figures 3C and 3D) was added to the medium at 0.1 mM. However, other amino acids such as L-alanine, L-aspartate, L-valine and L-glutamate had no effect on POA activity (Figures 3E–3H). This finding suggests that pantothenate and β-alanine, which are the downstream products of PanD, specifically antagonize the antituberculosis activity of POA and that POA inhibits pantothenate or β-alanine synthesis by blocking PanD activity in M. tuberculosis.


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)

Antagonism of POA's activity against M. tuberculosis by pantothenate and β-alanine. (A) M. tuberculosis parent strain H37Ra on 7H11 agar, (B) a POA-resistant mutant with a PanD mutation (M117I), (C) parent strain on 7H11 agar containing 0.1 mM calcium pantothenate, (D) parent strain with 0.1 mM β-alanine, (E) parent strain with 0.1 mM L-alanine, (F) parent strain with 5 mM calcium L-aspartate, (G) parent strain with 10 mM L-valine, (H) parent strain with 10 mM glutamate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Antagonism of POA's activity against M. tuberculosis by pantothenate and β-alanine. (A) M. tuberculosis parent strain H37Ra on 7H11 agar, (B) a POA-resistant mutant with a PanD mutation (M117I), (C) parent strain on 7H11 agar containing 0.1 mM calcium pantothenate, (D) parent strain with 0.1 mM β-alanine, (E) parent strain with 0.1 mM L-alanine, (F) parent strain with 5 mM calcium L-aspartate, (G) parent strain with 10 mM L-valine, (H) parent strain with 10 mM glutamate.
Mentions: Because PanD (aspartate decarboxylase) is involved in converting L-aspartic acid to β-alanine, a precursor for pantothenate and coenzyme A biosynthesis, we wondered if supplementation with β-alanine or pantothenate might render M. tuberculosis resistant to POA. To test this, pantothenate, β-alanine, and different amino acids that served as controls, were incorporated into 7H11 agar containing different concentrations of POA (200–800 µg/mL pH 6.8) followed by inoculation with M. tuberculosis H37Ra. The results showed that the parent strain and the mutant strain were sensitive and resistant to POA, respectively, on 7H11 plates containing different concentrations of POA (Figures 3A and 3B), but susceptibility to POA dramatically decreased when pantothenate or β-alanine (Figures 3C and 3D) was added to the medium at 0.1 mM. However, other amino acids such as L-alanine, L-aspartate, L-valine and L-glutamate had no effect on POA activity (Figures 3E–3H). This finding suggests that pantothenate and β-alanine, which are the downstream products of PanD, specifically antagonize the antituberculosis activity of POA and that POA inhibits pantothenate or β-alanine synthesis by blocking PanD activity in M. tuberculosis.

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