title: Mycobacterium tuberculosis dihydrofolate reductase is not a target relevant to the antitubercular activity of isoniazid.
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 INH-resistant 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.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Paras
authors.initials: P
authors.lastname: Jain
authors.forename: Gulcin
authors.initials: G
authors.lastname: Gulten
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
authors.forename: Yicheng
authors.initials: Y
authors.lastname: Feng
authors.forename: Krishna
authors.initials: K
authors.lastname: Ganesula
authors.forename: Alifiya S
authors.initials: AS
authors.lastname: Motiwala
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Ioerger
authors.forename: David
authors.initials: D
authors.lastname: Alland
authors.forename: Catherine
authors.initials: C
authors.lastname: Vilchèze
authors.forename: William R
authors.initials: WR
authors.lastname: Jacobs
authors.forename: James C
authors.initials: JC
authors.lastname: Sacchettini
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000502:physiology*; D014170:Transformation, Genetic / Q000235:genetics
chemical_list: D000995:Antitubercular Agents; D013762:Tetrahydrofolate Dehydrogenase; D007538:Isoniazid