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.