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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

M. tuberculosis became resistant to POA when panD expression was induced by ATc. Each strain was printed in triplicate with a replicator at a density of ∼105 CFU/mL. (A) WT/pUV15tetORs-panDRv (expressing panD from M. tuberculosis H37Rv) without (− sign) and with (+ sign) ATc, (B) WT/pUV15tetORs-panDG351A (expressing panD mutant) without and with ATc, (C) WT/pUV15tetORs-panDT431C (expressing panD mutant) without and with ATc, (D) POAR 156 (M117I) without and with ATc, (E) WT/pUV15tetORs-panDMS (expressing panD from M. smegmatis) without and with ATc, (F) WT/pUV15tetORs-panDEC (expressing panD from E. coli) without and with ATc, (G) WT/pUV15tetORs vector control without and with ATc.
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fig2: M. tuberculosis became resistant to POA when panD expression was induced by ATc. Each strain was printed in triplicate with a replicator at a density of ∼105 CFU/mL. (A) WT/pUV15tetORs-panDRv (expressing panD from M. tuberculosis H37Rv) without (− sign) and with (+ sign) ATc, (B) WT/pUV15tetORs-panDG351A (expressing panD mutant) without and with ATc, (C) WT/pUV15tetORs-panDT431C (expressing panD mutant) without and with ATc, (D) POAR 156 (M117I) without and with ATc, (E) WT/pUV15tetORs-panDMS (expressing panD from M. smegmatis) without and with ATc, (F) WT/pUV15tetORs-panDEC (expressing panD from E. coli) without and with ATc, (G) WT/pUV15tetORs vector control without and with ATc.

Mentions: To confirm PanD as a target of PZA, we overexpressed panD genes from M. tuberculosis in M. tuberculosis strain H37Ra and tested their PZA and POA susceptibility. Overexpression of the panD gene from wild-type M. tuberculosis, mutant POAR54 (nucleotide change G351A; amino acid change M117I), mutant POAR140 (nucleotide change T431C; amino acid change V138A), or the panD genes from M. smegmatis or E. coli caused POA resistance in M. tuberculosis H37Ra using the tetracycline-inducible vector pUV15tetORs14 in the presence of ATc (50 ng/mL) inducing agent (Figure 2). In contrast, the recombinant strains were sensitive to POA (<300 µg/mL, pH 6.8) without the inducing agent ATc (Figures 2A–2C). POA-resistant M. tuberculosis mutants with the M117I mutation grew in the absence or presence of ATc induction (Figure 2D) because this is the mutant alone and is not transformed with a panD expression construct. It is interesting to note that expression of either wild-type H37Rv panD or mutant panD (G351A, M117I and T431C, V138A) from M. tuberculosis conferred POA resistance to M. tuberculosis (POA>700 µg/mL, pH 6.8) (Figures 2B and 2C), as did panD from M. smegmatis and E. coli (Figures 2E and 2F),. The vector control remained susceptible to POA with or without ATc induction (Figure 2G). In addition, conditional overexpression of the above constructs caused a three-fold increase in the MIC of PZA for M. tuberculosis from 25 µg/mL in the uninduced and control strains to 75 µg/mL (pH 5.7) in the ATc-induced strains.


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)

M. tuberculosis became resistant to POA when panD expression was induced by ATc. Each strain was printed in triplicate with a replicator at a density of ∼105 CFU/mL. (A) WT/pUV15tetORs-panDRv (expressing panD from M. tuberculosis H37Rv) without (− sign) and with (+ sign) ATc, (B) WT/pUV15tetORs-panDG351A (expressing panD mutant) without and with ATc, (C) WT/pUV15tetORs-panDT431C (expressing panD mutant) without and with ATc, (D) POAR 156 (M117I) without and with ATc, (E) WT/pUV15tetORs-panDMS (expressing panD from M. smegmatis) without and with ATc, (F) WT/pUV15tetORs-panDEC (expressing panD from E. coli) without and with ATc, (G) WT/pUV15tetORs vector control without and with ATc.
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fig2: M. tuberculosis became resistant to POA when panD expression was induced by ATc. Each strain was printed in triplicate with a replicator at a density of ∼105 CFU/mL. (A) WT/pUV15tetORs-panDRv (expressing panD from M. tuberculosis H37Rv) without (− sign) and with (+ sign) ATc, (B) WT/pUV15tetORs-panDG351A (expressing panD mutant) without and with ATc, (C) WT/pUV15tetORs-panDT431C (expressing panD mutant) without and with ATc, (D) POAR 156 (M117I) without and with ATc, (E) WT/pUV15tetORs-panDMS (expressing panD from M. smegmatis) without and with ATc, (F) WT/pUV15tetORs-panDEC (expressing panD from E. coli) without and with ATc, (G) WT/pUV15tetORs vector control without and with ATc.
Mentions: To confirm PanD as a target of PZA, we overexpressed panD genes from M. tuberculosis in M. tuberculosis strain H37Ra and tested their PZA and POA susceptibility. Overexpression of the panD gene from wild-type M. tuberculosis, mutant POAR54 (nucleotide change G351A; amino acid change M117I), mutant POAR140 (nucleotide change T431C; amino acid change V138A), or the panD genes from M. smegmatis or E. coli caused POA resistance in M. tuberculosis H37Ra using the tetracycline-inducible vector pUV15tetORs14 in the presence of ATc (50 ng/mL) inducing agent (Figure 2). In contrast, the recombinant strains were sensitive to POA (<300 µg/mL, pH 6.8) without the inducing agent ATc (Figures 2A–2C). POA-resistant M. tuberculosis mutants with the M117I mutation grew in the absence or presence of ATc induction (Figure 2D) because this is the mutant alone and is not transformed with a panD expression construct. It is interesting to note that expression of either wild-type H37Rv panD or mutant panD (G351A, M117I and T431C, V138A) from M. tuberculosis conferred POA resistance to M. tuberculosis (POA>700 µg/mL, pH 6.8) (Figures 2B and 2C), as did panD from M. smegmatis and E. coli (Figures 2E and 2F),. The vector control remained susceptible to POA with or without ATc induction (Figure 2G). In addition, conditional overexpression of the above constructs caused a three-fold increase in the MIC of PZA for M. tuberculosis from 25 µg/mL in the uninduced and control strains to 75 µg/mL (pH 5.7) in the ATc-induced strains.

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