Mycobacterium tuberculosis dihydrofolate reductase is not a target relevant to the antitubercular activity of isoniazid

Feng Wang, Paras Jain, Gulcin Gulten, Zhen Liu, Yicheng Feng, Krishna Ganesula, Alifiya S. Motiwala, Thomas R. Ioerger, David Alland, Catherine Vilchèze, William R. Jacobs, James C. Sacchettini

Research output: Contribution to journalArticle

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Abstract

Mycobacterium tuberculosis enoyl-acyl-ACP reductase (InhA) has been demonstrated to be the primary target of isoniazid (INH). Recently, it was postulated that M. tuberculosis dihydrofolate reductase (DHFR) is also a target of INH, based on the findings that a 4R-INH-NADP adduct synthesized from INH by a nonenzymatic approach showed strong inhibition of DHFR in vitro, and overexpression of M. tuberculosis dfrA in M. smegmatis conferred a 2-fold increase of resistance to INH. In the present study, a plasmid expressing M. tuberculosis dfrA was transformed into M. smegmatis and M. tuberculosis strains, respectively. The transformant strains were tested for their resistance to INH. Compared to the wild-type strains, overexpression of dfrA in M. smegmatis and M. tuberculosis did not confer any resistance to INH based on the MIC values. Similar negative results were obtained with 14 other overexpressed proteins that have been proposed to bind some form of INH-NAD(P) adduct. An Escherichia coli cell-based system was designed that allowed coexpression of both M. tuberculosis katG and dfrA genes in the presence of INH. The DHFR protein isolated from the experimental sample was not found bound with any INH-NADP adduct by enzyme inhibition assay and mass spectroscopic analysis. We also used whole-genome sequencing to determine whether polymorphisms in dfrA could be detected in six INHresistant clinical isolates known to lack mutations in inhA and katG, but no such mutations were found. The dfrA overexpression experiments, together with the biochemical and sequencing studies, conclusively demonstrate that DHFR is not a target relevant to the antitubercular activity of INH.

Original languageEnglish (US)
Pages (from-to)3776-3782
Number of pages7
JournalAntimicrobial Agents and Chemotherapy
Volume54
Issue number9
DOIs
StatePublished - Sep 2010

Fingerprint

Tetrahydrofolate Dehydrogenase
Isoniazid
Mycobacterium tuberculosis
Smegma
NADP
Mutation
Enzyme Assays
Oxidoreductases
Proteins
Plasmids
Genome
Escherichia coli
Genes

ASJC Scopus subject areas

  • Pharmacology (medical)
  • Pharmacology
  • Infectious Diseases

Cite this

Mycobacterium tuberculosis dihydrofolate reductase is not a target relevant to the antitubercular activity of isoniazid. / Wang, Feng; Jain, Paras; Gulten, Gulcin; Liu, Zhen; Feng, Yicheng; Ganesula, Krishna; Motiwala, Alifiya S.; Ioerger, Thomas R.; Alland, David; Vilchèze, Catherine; Jacobs, William R.; Sacchettini, James C.

In: Antimicrobial Agents and Chemotherapy, Vol. 54, No. 9, 09.2010, p. 3776-3782.

Research output: Contribution to journalArticle

Wang, F, Jain, P, Gulten, G, Liu, Z, Feng, Y, Ganesula, K, Motiwala, AS, Ioerger, TR, Alland, D, Vilchèze, C, Jacobs, WR & Sacchettini, JC 2010, 'Mycobacterium tuberculosis dihydrofolate reductase is not a target relevant to the antitubercular activity of isoniazid', Antimicrobial Agents and Chemotherapy, vol. 54, no. 9, pp. 3776-3782. https://doi.org/10.1128/AAC.00453-10
Wang, Feng ; Jain, Paras ; Gulten, Gulcin ; Liu, Zhen ; Feng, Yicheng ; Ganesula, Krishna ; Motiwala, Alifiya S. ; Ioerger, Thomas R. ; Alland, David ; Vilchèze, Catherine ; Jacobs, William R. ; Sacchettini, James C. / Mycobacterium tuberculosis dihydrofolate reductase is not a target relevant to the antitubercular activity of isoniazid. In: Antimicrobial Agents and Chemotherapy. 2010 ; Vol. 54, No. 9. pp. 3776-3782.
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