Transfer of a point mutation in
Mycobacterium tuberculosis inhA
resolves the target of isoniazid
Catherine Vilche `ze1, Feng Wang2, Masayoshi Arai1,
Manzour Hernando Hazbo ´n3, Roberto Colangeli3,
Laurent Kremer4, Torin R Weisbrod1, David Alland3,
James C Sacchettini2& William R Jacobs, Jr1
Isoniazid is one of the most effective antituberculosis drugs,
yet its precise mechanism of action is still controversial. Using
specialized linkage transduction, a single point mutation allele
(S94A) within the putative target gene inhA was transferred
in Mycobacterium tuberculosis. The inhA(S94A) allele was
sufficient to confer clinically relevant levels of resistance to
isoniazid killing and inhibition of mycolic acid biosynthesis.
This resistance correlated with the decreased binding of the
INH-NAD inhibitor to InhA, as shown by enzymatic and X-ray
crystallographic analyses, and establishes InhA as the primary
target of isoniazid action in M. tuberculosis.
Isoniazid (INH), whose bactericidal activity against Mycobacterium
tuberculosis was discovered in 1952 (ref. 1), has become the foundation
of modern chemotherapy for active and latent tuberculosis because of
its excellent activity, low cost and relatively low toxicity. The mode of
action of INH has been controversial, with early reports suggesting
that INH affected cell permeability, inhibited DNA biosynthesis,
altered NAD metabolism or inhibited mycolic acid biosynthesis. The
mycolic acid biosynthesis inhibition hypothesis was validated by the
demonstration that INH-induced inhibition of mycolic acid biosynth-
esis correlated with cell death2, and the putative target of INH was
postulated in 1975 to be a desaturase, a cyclopropanase or an enzyme
involved in fatty-acid elongation3.
The development of plasmid genomic library transfer systems for
mycobacteria in 1994 (ref. 4) and the completion of the M. tuberculosis
genome sequence in 1998 (ref. 5) spurred further progress. By trans-
ferring M. tuberculosis genes on multicopy plasmids in M. smegmatis,
the inhA gene, encoding an NADH-specific enoyl-acyl carrier protein
(ACP) reductase, was identified in 1994 as a putative target for both
INH and the related drug ethionamide (ETH)4. The b-ketoacyl ACP
synthase (KasA)6and, more recently, the dihydrofolate reductase
(DHFR)7have also been proposed as targets of INH, based on INH
binding studies. Moreover, mutations in inhA and kasA have also been
found in M. tuberculosis INH-resistant clinical isolates6,8. The
determination of a clinically relevant drug target, however, requires the
ability to transfer such a single point mutation within a gene that
putatively encodes a drug target and demonstrate that this transfer is
sufficient, by itself, to confer drug resistance.
We performed a specialized linkage transduction9to introduce the
inhA(S94A) point mutation, previously associated with INH resis-
tance4, linked to a gene conferring hygromycin resistance, into
M. tuberculosis (Fig. 1a) or M. bovis BCG (Supplementary Methods
online). Two different types of hygromycin-resistant recombinants
could be obtained, depending on the site of recombination with respect
to the S94A mutation, resulting in INH resistance or susceptibility
(Fig. 1b). In M. tuberculosis, 51% of the hygromycin-resistant trans-
ductants were INH resistant (Fig. 1c). We screened the transductants
for the absence or presence of the inhA(S94A) allele using sequence
analysis or a hairpin-shaped primer assay and found a 100% correla-
tion with INH resistance, ETH resistance and the presence of the
inhA(S94A) allele. As predicted, the introduction of the S94A mutation
was sufficient for conferring at least a fivefold resistance to INH and
ETH in M. tuberculosis and M. bovis BCG (Supplementary Table 1 and
Supplementary Fig. 1 online). We also introduced three kasA muta-
tions (G269S, G312S and F413L), found in INH-resistant M. tubercu-
losis clinical isolates6, into M. tuberculosis or M. bovis BCG using
specialized linkage transduction to test whether these mutations con-
ferred INH resistance. The presence of mutated kasA alleles was
confirmed by sequencing. None of the transductants obtained showed
any detectable level of resistance to INH (Supplementary Table 1
online). These results indicate that no single kasA mutation was
sufficient, by itself, to confer INH resistance in M. tuberculosis.
The bactericidal activity of INH correlates with the inhibition of
mycolic acid biosynthesis2. Therefore, we analyzed two inhA isogenic
strains carrying either the wild-type inhA allele (mc24910) or the
inhA(S94A) allele (mc24911) for their resistance to mycolic acid
biosynthesis inhibition in the presence of increasing concentrations
of INH. At 0.25 mg/ml INH, mycolic acid biosynthesis was fully
inhibited in INH-resistant mc24910, but not in INH-resistant mc24911
(Fig. 1d). Complete inhibition of mycolic acid biosynthesis in both
M. tuberculosis isogenic strains occurred at twofold the minimum
inhibitory concentration (MIC), in agreement with the bacteriocidal
As INH resistance can be mediated by overexpression of inhA10, we
compared the inhA mRNA levels between mc24910 and mc24911 at
different concentrations of INH, using a molecular beacon RT-PCR
assay. The mRNA levels were similar in both strains even at high
concentrations of INH (Fig. 1e), and this corresponded to InhA
protein levels (Fig. 1f). Therefore, INH resistance in mc24911 was not
caused by inhAoverexpression. In contrast, mc24914, an INH-resistant
Received 16 March; accepted 11 July; published online 13 August 2006; doi:10.1038/nm1466
1Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York
10461, USA.2Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.3Division of Infectious Disease, Department of
Medicine and the Ruy V. Lourenc ¸o Center for the Study of Emerging and Re-emerging Pathogens, New Jersey Medical School, University of Medicine and Dentistry of
New Jersey, 185 South Orange Avenue, MSB A920B, Newark, New Jersey 07103, USA.4Laboratoire de Dynamique Mole ´culaire des Interactions Membranaires, CNRS
UMR 5539, Universite ´ de Montpellier II, case 107, Place Euge `ne Bataillon, 34095 Montpellier Cedex 05, France. Correspondence should be addressed to W.R.J.
VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2006 1027
© 2006 Nature Publishing Group http://www.nature.com/naturemedicine
M. tuberculosis spontaneous mutant carrying the inhA expression
region mutation (C–15T), which had been proposed as inducing
overexpression of inhA, showed a 20-fold increase in inhA mRNA
levels (Fig. 1e) and subsequent InhA protein levels (Fig. 1f). It
has been suggested that this mutation maps to the ribosomal initia-
tion site, so it was unclear whether it affected transcription or
translation. This analysis shows that the C–15T inhA promoter
mutation mediates enhanced transcription of inhA mRNA levels,
resulting in INH resistance.
It was previously shown that kasA is induced in mycobacteria
treated with INH6. In agreement, we found that M. tuberculosis strains
H37Rv and mc24910 have increased kasA mRNA levels when treated
with INH (Fig. 1e). In contrast, mc24911 and mc24914, which are
resistant to INH, showed no increase in kasA mRNA levels when
treated with INH at or below the MIC. An increase in the level of kasA
mRNA was observed in mc24911 at 1.0 mg/ml INH. This suggests that
kasA mRNA levels were induced only when InhA was inhibited, and
that the increase in kasA expression does not correlate with INH
resistance, a fact that is consistent with the observation that kasA
overexpression in fast- and slow-growing mycobacteria does not
increase resistance to INH10.
It has been shown that InhA is inhibited by a covalent INH-NAD
adduct11, the product of activated INH with NAD+(ref. 12). To
determine whether the InhA(S94A) enzyme is resistant to inhibition
by the INH-NAD adduct, we measured the inactivation of wild-type
and mutated InhA by this adduct in a dose-dependent fashion. The
InhA(S94A) enzyme was 17 times more resistant to inhibition by
the INH-NAD adduct (IC50, 323 ± 41 nM for the InhA(S94A)
enzyme versus 19 ± 10 nM for the wild-type enzyme), and showed a
30-fold increase in the Kivalue for the INH-NAD adduct (Kifor the
S94A mutant protein is 172 ± 22 nM; Kifor the wild-type protein is 5 ±
3 nM). To determine the molecular basis for the reduced inhibition of
the INH-NAD adduct to the InhA(S94A) enzyme, we co-crystallized
the InhA(S94A) protein with the INH-NAD adduct (Fig. 2a)
and compared it to the wild-type protein structure. Although the
position and orientation of the INH-NAD adduct was nearly identical
in the active sites of the wild-type (Fig. 2b) and InhA(S94A)
(Fig. 2c) proteins, the loss of the serine residue resulted in
the movement of an ordered water molecule that disrupted the
hydrogen bonding network, which probably decreases the binding of
the adduct to the InhA(S94A) protein.
Elucidating the mechanism of action of INH has been a complex
endeavor because of: (i) the prior inability to transfer point mutations
in M. tuberculosis; (ii) the clinically irrelevant binding of INH to
proteins6,7; (iii) the fact that INH is a prodrug that requires modifica-
tion before becoming active13; (iv) the 100-fold difference in suscept-
ibility between M. smegmatis and M. tuberculosis; and (v) the initial
inability to explain the accumulation of saturated C26fatty acid upon
INH treatment14. Although a drug may bind to a number of different
enzymes in vitro, the validation of a proposed target requires in vivo
data. In vivo binding can be established by identifying amino acid
substitutions within the target enzyme that reduce binding or by
mabA inhA(S94A) hyg hemZ
M. tb chromosome
Allelic exchange - Type 1
Allelic exchange - Type 2
INH (µg/ml): 0 0.06 0.12 0.25 0.5 1.0 0 0.06 0.12 0.25 0.5 1.0
H37Rv mc24911 mc24910 mc24914H37Rv mc24911 mc24910 mc24914
0.2 1.00 0.2 1.000.2 1.0 0 0.2 1.0
Figure 1 Construction and analysis of M. tuberculosis inhA(S94A) (Supplementary Methods). (a) Schematic representation of the specialized transducing
phage. A replicating shuttle phasmid phAE2067 containing mabA, inhA carrying the S94A mutation, a hyg resistance cassette and hemZ was used to
transduce M. tuberculosis (M. tb). The two possible sites of recombination are marked 1 and 2. (b) The recombination can occur either before the point
mutation (crossover type 1), resulting in an INH-resistant and ETH-resistant recombinant carrying the S94A mutation, or after the point mutation (crossover
type 2; the strain contains a wild-type inhA gene). (c) Individual M. tuberculosis H37Rv inhA(S94A) transductants (n ¼ 150) were screened by picking and
patching onto plates containing either hygromycin (50 mg/ml) or INH (0.2 mg/ml). (d) Fatty acid methyl esters (FAMEs) and mycolic acid methyl esters
(MAMEs) profiles of mc24910 (INH-sensitive) and mc24911 (INH-resistant). The M. tuberculosis strains were treated with different concentrations of INH
for 4 h and labeled with [1-14C]-acetate.14C-labeled FAMEs and MAMEs were separated by thin-layer chromatography and detected by autoradiography.
(e) Analysis of inhA and kasA mRNA levels of M. tuberculosis strains by RT-PCR. The M. tuberculosis cultures were treated with INH for 4 h before measuring
the inhA and kasA mRNA levels. inhA and kasA values were normalized using sigA levels. Means (n ¼ 3) ± s.e.m. (*P o 0.05). (f) Western blot analysis of
InhA protein levels of M. tuberculosis strains treated with INH for 4 h. InhA was detected in total protein extract using rabbit antibodies to InhA raised against
the M. tuberculosis InhA protein.
1028VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2006
© 2006 Nature Publishing Group http://www.nature.com/naturemedicine
showing target overexpression, both of which confer clinically relevant Download full-text
The coupling of specialized linkage transduction with enzymatic
analyses and X-ray crystallography has enabled us to establish the pre-
cise molecular mechanism by which INH inhibits InhA, and supports
the premise that inhA encodes the primary target of INH. Further-
more, the observations that inhA modification or overexpression10, or
altered NADH/NAD ratios15, confers co-resistance to ETH support
the hypothesis that InhA is a common target of both INH and ETH.
In addition to identifying the actual drug target of INH, the INH
story has unveiled a new paradigm of drug action, in which
INH functions by covalently modifying an enzyme cofactor. This
paradigm provides important insights that may be valuable in devel-
oping new drugs against multidrug resistant and extensively drug-
resistant M. tuberculosis (http://www.cdc.gov) as well as against other
pathogenic mycobacteria like M. ulcerans and M. leprae.
Note: Supplementary information is available on the Nature Medicine website.
We gratefully acknowledge G. Morlock for sending us the DNA from the
INH-resistant clinical isolate carrying the inhA(S94A) mutation. L. Kremer is
supported by a grant from the Centre National de la Recherche Scientifique
(ATIP ‘‘Microbiologie Fondamentale’’). J.C.S. acknowledges the Robert A. Welch
Foundation grant A-0015. We also acknowledge support for this work from
US National Institutes of Health grants AI43268 and AI46669, and from the
Structural Genomics Project grant 1P50GM6241. We also thank G. Hatfull
for careful reading of this manuscript.
C.V. performed specialized transductions of inhA in M. tuberculosis and BCG
and analyzed the transductants for their inserts, INH and ETH resistance and
biochemical resistance to INH, and wrote most of the manuscript with W.R.J.
F.W. prepared the INH-NAD adduct, performed the InhA enzymological analyses
and X-ray crystallography. M.A. constructed the phages for transduction and
performed and analyzed the kasA allelic exchanges in M. tuberculosis and
M. bovis BCG. T.R.W. sequenced the transductants. M.H.H. and R.C. conducted
the hairpin-shaped primer assays and mRNA experiments. L.K. provided
the pMV261::kasA constructs and did the western blot analysis. D.A., J.C.S.
and W.R.J. contributed to the design of the study, data analysis and
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
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Figure 2 X-ray crystallographic analysis of the INH-NAD adduct bound to wild-type InhA or InhA(S94A) protein. (a) Crystal structure of the S94A mutant
protein with the INH-NAD adduct. (b) In the InhA–INH-NAD structure, the oxygen O9 of the phosphate of the INH-NAD adduct forms one hydrogen bond
with the main-chain nitrogen atom of Ile21 and one hydrogen bond with a well-ordered water molecule. This water molecule is part of a hydrogen-bonding
network formed by interactions between the side-chain oxygen atom of Ser94 (2.85 A˚), the main-chain oxygen of Gly14 (2.78 A˚) and the oxygen atoms O3
(3.26 A˚) and O9 (2.82 A˚) of the INH-NAD adduct. The same water molecule is within hydrogen-bonding distance of the main-chain nitrogen atoms of Ala22
(2.9 A˚) and Ile21 (3.3 A˚). (c) In the InhA(S94A)–INH-NAD structure, this hydrogen-bonding network is disrupted by the loss of the hydroxyl group in the
S94A substitution. Although the water molecule is still visible in the same position, it is 2.92 A˚from the main-chain oxygen of Gly14 and out of range of
hydrogen bonding interaction with the oxygen atom O3 (3.54 A˚). The water molecule is 0.2 A˚and 0.1 A˚closer to the adjacent residues Ala22 and Ile21,
which form hydrogen bonds with their main-chain nitrogen atoms, respectively.
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