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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

Overexpression and purification of M. tuberculosis PanD in E. coli. Lane 1–4: PanD protein, which appears as a 16-kD band, eluted by 50, 100, 200, 400 mM imidazole, respectively. Lane 5: Protein molecular weight marker (116, 66, 45, 35, 25, 18, 14 kD).
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fig6: Overexpression and purification of M. tuberculosis PanD in E. coli. Lane 1–4: PanD protein, which appears as a 16-kD band, eluted by 50, 100, 200, 400 mM imidazole, respectively. Lane 5: Protein molecular weight marker (116, 66, 45, 35, 25, 18, 14 kD).

Mentions: PanD (aspartate decarboxylase) catalyzes the formation of β-alanine from aspartic acid in the reaction L-aspartate + H+ → β-alanine + CO2. To assess if POA can inhibit the enzymatic activity of PanD, we overexpressed and purified the wild-type M. tuberculosis PanD protein, which appeared as a 16 kD band on SDS-PAGE (Figure 6). The purified PanD protein had activity of 107.8 U/mg and was used for a PanD enzymatic assay in the presence or absence of POA. The PanD M117I mutant was similarly overexpressed and purified and was found to have slightly reduced enzymatic activity (76.3 U/mg). The wild-type M. tuberculosis PanD hydrolyzed aspartic acid (Asp) to β-alanine (β-Ala) in the absence of POA (Figure 7A, Lane 4 from the left). Importantly, POA at the physiologically relevant concentrations of 100, 50, or 25 µg/mL inhibited the activity of M. tuberculosis PanD in a concentration-dependent manner with 23%, 27%, or 39% residual enzymatic activity as shown by the increasing accumulation of the substrate aspartate and the decreasing amount of the product β-alanine (Figure 7A). The M117I PanD mutant enzyme was also inhibited by POA (100, 50, 25 µg/mL) to varying degrees with 16%, 32%, and 50% residual enzymatic activity, except that the M117I mutant enzyme appeared to be slightly less inhibited by POA than the wild-type enzyme was at 50 and 25 µg/mL (Figure 7A). POA at 200 µg/mL completely inhibited the activity of the wild-type PanD enzyme as shown by the lack of conversion of aspartate to β-alanine (Figures 7B and 7C, first lane from left). In contrast, neither the prodrug PZA nor nicotinamide when used as controls at 100 or 200 µg/mL inhibited the enzymatic activity of PanD (Figures 7B and 7C) as expected.


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)

Overexpression and purification of M. tuberculosis PanD in E. coli. Lane 1–4: PanD protein, which appears as a 16-kD band, eluted by 50, 100, 200, 400 mM imidazole, respectively. Lane 5: Protein molecular weight marker (116, 66, 45, 35, 25, 18, 14 kD).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Overexpression and purification of M. tuberculosis PanD in E. coli. Lane 1–4: PanD protein, which appears as a 16-kD band, eluted by 50, 100, 200, 400 mM imidazole, respectively. Lane 5: Protein molecular weight marker (116, 66, 45, 35, 25, 18, 14 kD).
Mentions: PanD (aspartate decarboxylase) catalyzes the formation of β-alanine from aspartic acid in the reaction L-aspartate + H+ → β-alanine + CO2. To assess if POA can inhibit the enzymatic activity of PanD, we overexpressed and purified the wild-type M. tuberculosis PanD protein, which appeared as a 16 kD band on SDS-PAGE (Figure 6). The purified PanD protein had activity of 107.8 U/mg and was used for a PanD enzymatic assay in the presence or absence of POA. The PanD M117I mutant was similarly overexpressed and purified and was found to have slightly reduced enzymatic activity (76.3 U/mg). The wild-type M. tuberculosis PanD hydrolyzed aspartic acid (Asp) to β-alanine (β-Ala) in the absence of POA (Figure 7A, Lane 4 from the left). Importantly, POA at the physiologically relevant concentrations of 100, 50, or 25 µg/mL inhibited the activity of M. tuberculosis PanD in a concentration-dependent manner with 23%, 27%, or 39% residual enzymatic activity as shown by the increasing accumulation of the substrate aspartate and the decreasing amount of the product β-alanine (Figure 7A). The M117I PanD mutant enzyme was also inhibited by POA (100, 50, 25 µg/mL) to varying degrees with 16%, 32%, and 50% residual enzymatic activity, except that the M117I mutant enzyme appeared to be slightly less inhibited by POA than the wild-type enzyme was at 50 and 25 µg/mL (Figure 7A). POA at 200 µg/mL completely inhibited the activity of the wild-type PanD enzyme as shown by the lack of conversion of aspartate to β-alanine (Figures 7B and 7C, first lane from left). In contrast, neither the prodrug PZA nor nicotinamide when used as controls at 100 or 200 µg/mL inhibited the enzymatic activity of PanD (Figures 7B and 7C) as expected.

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