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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

Alignment of the PanD amino acid sequences of M. tuberculosis (MTB), M. smegmatis (MSG) and E. coli (ECO). The conserved residues are boxed. The C-terminal sequence without crystal structure is highlighted in yellow. The asterisks indicate the mutant residues identified in the POA-resistant M. tuberculosis mutants.
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fig1: Alignment of the PanD amino acid sequences of M. tuberculosis (MTB), M. smegmatis (MSG) and E. coli (ECO). The conserved residues are boxed. The C-terminal sequence without crystal structure is highlighted in yellow. The asterisks indicate the mutant residues identified in the POA-resistant M. tuberculosis mutants.

Mentions: To identify possible new targets of POA, we plated M. tuberculosis H37Ra on 7H11 plates containing POA (25–200 µg/mL at pH 5.7 or 200–700 µg/mL at pH 6.8). Through two more rounds of screening, we were able to isolate 30 mutants resistant to both POA and PZA. DNA sequencing revealed that these 30 POA-resistant mutants did not have the mutations in pncA and rpsA known to cause PZA resistance. Surprisingly, whole-genome sequencing analysis of 3 POA-resistant mutants revealed they had various point mutations (M117I, C deletion at nucleotide position 393, V138A mutations) in the panD gene (Table 1). In view of this finding, the remaining 27 POA-resistant mutants were all subjected to panD DNA sequencing, and remarkably, the results showed that they all had PanD mutations (Table 1). Interestingly, all the mutations occurred in the C-terminus of the 139 amino acid long M. tuberculosis PanD protein within amino acid residues 117–138 (Figure 1). When compared with the wild-type PanD protein, the majority of the mutants (24 of 30 or 80%) had the M117I mutation, and the remaining 6 had the mutations L136R, V138G, V138E, V138A, E126* and a codon shift at 131 (Table 1 and Figure 1). Sequence alignment of the PanD proteins from M. tuberculosis, M. smegmatis, and E. coli indicates that the C-terminal region of M. tuberculosis PanD where all the PanD mutations occurred is variable and confers sequence specificity (Figure 1).


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)

Alignment of the PanD amino acid sequences of M. tuberculosis (MTB), M. smegmatis (MSG) and E. coli (ECO). The conserved residues are boxed. The C-terminal sequence without crystal structure is highlighted in yellow. The asterisks indicate the mutant residues identified in the POA-resistant M. tuberculosis mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Alignment of the PanD amino acid sequences of M. tuberculosis (MTB), M. smegmatis (MSG) and E. coli (ECO). The conserved residues are boxed. The C-terminal sequence without crystal structure is highlighted in yellow. The asterisks indicate the mutant residues identified in the POA-resistant M. tuberculosis mutants.
Mentions: To identify possible new targets of POA, we plated M. tuberculosis H37Ra on 7H11 plates containing POA (25–200 µg/mL at pH 5.7 or 200–700 µg/mL at pH 6.8). Through two more rounds of screening, we were able to isolate 30 mutants resistant to both POA and PZA. DNA sequencing revealed that these 30 POA-resistant mutants did not have the mutations in pncA and rpsA known to cause PZA resistance. Surprisingly, whole-genome sequencing analysis of 3 POA-resistant mutants revealed they had various point mutations (M117I, C deletion at nucleotide position 393, V138A mutations) in the panD gene (Table 1). In view of this finding, the remaining 27 POA-resistant mutants were all subjected to panD DNA sequencing, and remarkably, the results showed that they all had PanD mutations (Table 1). Interestingly, all the mutations occurred in the C-terminus of the 139 amino acid long M. tuberculosis PanD protein within amino acid residues 117–138 (Figure 1). When compared with the wild-type PanD protein, the majority of the mutants (24 of 30 or 80%) had the M117I mutation, and the remaining 6 had the mutations L136R, V138G, V138E, V138A, E126* and a codon shift at 131 (Table 1 and Figure 1). Sequence alignment of the PanD proteins from M. tuberculosis, M. smegmatis, and E. coli indicates that the C-terminal region of M. tuberculosis PanD where all the PanD mutations occurred is variable and confers sequence specificity (Figure 1).

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