A gene (nat) encoding arylamine N-acetyltransferase (NAT) has been found in Mycobacterium tuberculosis. The gene is expressed and the enzyme is active in growing M. tuberculosis cells. N-Acetyltransferase acetylates and inactivates isoniazid (INH), which is a front-line drug used in tuberculosis (TB) therapy. In this study, it was shown that a previously reported G619A single nucleotide polymorphism (SNP) was conserved in two M. tuberculosis strain families found in the Western Cape Province of South Africa (strain families 3 and 28). Further sequence analysis of isolates in strain family 3 identified a new T529C SNP in NAT resulting in a histidine instead of a tyrosine at position 177. This SNP was found only in isolates from strain family 3, and this mutation affects the highly conserved tyrosine residue close to the active site. Using real-time PCR, the expression of M. tuberculosis nat (tbnat) was determined over a 28 day growth cycle of the M. tuberculosis reference strain (H37Rv). The expression of tbnat occurs early in growth and reaches maximum levels at mid-exponential phase. The exposure of INH-susceptible isolates to low levels of INH resulted in an increase of tbnat expression (reference strain H37Rv, which is wild-type for tbnat, and isolate 1430, containing both SNPs). An INH-resistant isolate (816) exposed to INH showed no change in tbnat expression. The increased expression in the susceptible isolates suggests that INH affects tbnat expression. tbnat may contribute to INH susceptibility, but in combination with other factors.
AllandD.,
KramnikI.,
WeisbrodT. R.,
OtsuboL.,
CernyR.,
MillerL. P.,
JacobsR. F.Jr,
BloomB. R.1998; Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL) : the effect of INH on gene expression in Mycobacterium tuberculosis
. Proc Natl Acad Sci U S A 95:13227–13232[CrossRef]
AndertonM. C.,
BhaktaS. Patrick.,
LA,
SimE.2004; Characterisation of the putative operon containing arylamine N -acetyltransferase (NAT) in mycobacteria. Drug Metab Rev 36:supplement 1 abstract 322
BanerjeeA.,
DubnauE.,
QuemardA.,
BalasubramanianV.,
Sun UmK.,
WilsonT.,
CollinsD.,
de LisleG.,
JacobsW. R.1994; inhA , a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis
. Science 263:227–232[CrossRef]
BhaktaS.,
BesraG. S.,
UptonA. & 8 other authors; 2004; Arylamine N- acetyltransferase is required for synthesis of mycolic acids and complex lipids in Mycobacterium bovis BCG and represents a novel drug target. J Exp Med 199:1191–1199[CrossRef]
CarmoM.,
PeiexoC.,
CoroadinhaA. S.,
AlvesP. M.,
CruzP. E.,
CarrondoM. J. T.2004; Quantitation of MLV-based retroviral vectors using real-time RT-PCR. J Virol Methods 119:115–119[CrossRef]
Gonzalez-y-MerchandJ. A.,
ColstonM. J.,
CoxR. A.1998; Roles of multiple promoters in transcription of ribosomal DNA: effects of growth conditions on precursor rRNA synthesis in mycobacteria. J Bacteriol 180:5756–5761
GutackerM. M.,
SmootJ. C.,
Lux MigliaccioC. A. & 7 other authors; 2002; Genome-wide analysis of synonymous single nucleotide polymorphisms in Mycobacterium tuberculosis complex organisms: resolution of genetic relationships among closely related microbial strains. Genetics 162:1533–1543
HansenM. C.,
NielsenA. K.,
MolinS.,
HammerK.,
KilstrupM.2001; Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels. J Bacteriol 183:4747–4751[CrossRef]
HellyerT. J.,
DesJardenL. E.,
HehmanG. L.,
CaveM. D.,
EisenachK. D.1999; Quantitative analysis of mRNA as a marker for viability of M.tuberculosis
. J Clin Microbiol 37:290–295
KawamuraA.,
SandyJ.,
UptonA.,
NobleM.,
SimE.2003; Structural investigation of mutant Mycobacterium smegmatis arylamine N -acetyltransferase: a model for a naturally occurring functional polymorphism in Mycobacterium tuberculosis arylamine N -acetyltransferase. Protein Expr Purif 27:75–84[CrossRef]
MdluliK.,
SlaydenR. A.,
ZhuY.,
RamaswamyS.,
PanX.,
MeadD.,
CraneD. D.,
MusserJ.,
BarryC. E. I.1998; Inhibition of a Mycobacterium tuberculosis β-ketoacyl ACP synthase by isoniazid. Science 280:1607–1610[CrossRef]
MeyersP. R.,
BournW. R.,
SteynL. M.,
van HeldenP. D.,
BeyersA. D.,
BrownG. D.1998; Novel method for rapid measurement of growth of mycobacteria in detergent-free media. J Clin Microbiol 36:2752–2754
MusserM.,
KapurV.,
WilliamsD.,
KreiswirthB.,
van SoolingenD.,
Van EmbdenJ.1996; Characterisation of the catalase-peroxidase gene ( katG ) and inhA locus in isoniazid resistant and susceptible strains of Mycobacterium tuberculosis by automated DNA sequencing: restricted array of mutations associated with drug resistance. J Infect Dis 173:196–202[CrossRef]
ParkinD. P.,
VandenplasS.,
BothaF. J.,
VandenplasM. L.,
SeifartH. I.,
van HeldenP. D.,
van der WaltB. J.,
DonaldP. R.,
van JaarsveldP. P.1997; Trimodality of isoniazid elimination: phenotype and genotype in patients with tuberculosis. Am J Respir Crit Care Med 155:1717–1722[CrossRef]
PaytonM.,
AutyR.,
DelgodaR.,
EverettM.,
SimE.1999; Cloning and characterization of arylamine N -acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis : increased expression results in isoniazid resistance. J Bacteriol 181:1343–1347
PaytonM.,
GiffordC.,
SchartauP.,
HagaemierC.,
MushtaqA.,
LucasS.,
PinterK.,
SimE.2001a; Evidence towards the role of arylamine N -acetyltransferase in Mycobacterium smegmatis and development of a specific antiserum against the homologous enzyme of Mycobacterium tuberculosis
. Microbiology 147:3295–3302
PaytonM.,
MushtaqA.,
YuT. W.,
WuL.-J.,
SinclairJ.,
SimE.2001b; Eubacterial arylamine N -acetyltransferases - identification and comparison of 18 members of the protein family with conserved active site cysteine, histidine and aspartate residues. Microbiology 147:1137–1147
SandyJ.,
MushtaqA.,
HoltonS. J.,
NobleM.,
SimE.2005; The catalytic triad of residues of arylamine N -acetyltransferase are all essential for catalytic activity. Biochem J 390:115–123[CrossRef]
SimE.,
PaytonM.,
NobleM.,
MinchinR.2000; An update on genetic, structural and functional studies of arylamine N -acetyltransferases in eucaryotes and procaryotes. Hum Mol Genet 9:2435–2441[CrossRef]
SlaydenR. A.,
BarryC. E.2000; The genetics and biochemistry of isoniazid resistance in Mycobacterium tuberculosis
. Microbes Infect 2:659–669[CrossRef]
TorresM. J.,
CriadoA.,
PalomaresJ. C.,
AznarJ.2000; Use of real-time PCR and fluorimetry for rapid detection of rifampin and isoniazid resistance-associated mutations in Mycobacterium tuberculosis
. J Clin Microbiol 38:3194–3199
UptonA.,
MushtaqA.,
VictorT.,
SampsonS. L.,
SandyJ.,
SmithD.-M.,
van HeldenP.,
SimE.2001; Arylamine N -acetyltransferase of Mycobacterium tuberculosis is a polymorphic enzyme and a site of isoniazid metabolism. Mol Microbiol 42:309–317[CrossRef]
VainrubA.,
Montgomery PetittB.2003; Surface electrostatic effects in oligonucleotide microarrays: control and optimization of binding thermodynamics. Biopolymers 68:265–270[CrossRef]
VandecasteeleS. J.,
PeetermansW. E.,
MerckR.,
van EldereJ.2001; Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. J Bacteriol 183:7094–7101[CrossRef]
van RieA.,
WarrenR.,
MshangaI. & 9 other authors; 2001; Analysis for a limited number of gene codons can predict drug resistance of Mycobacterium tuberculosis in a high-incidence community. J Clin Microbiol 39:636–641[CrossRef]
VictorT.,
WarrenR.,
BeyersN.,
van HeldenP.1997; Transmission of multidrug-resistant strains of Mycobacterium tuberculosis in a high incidence community. Eur J Clin Microbial Infect Dis 16:548–549[CrossRef]
VictorT. C.,
van HeldenP. D.,
WarrenR.2002; Prediction of drug resistance in M.tuberculosis : molecular mechanism, tools, and applications. IUBMB Life 53:231–237[CrossRef]
WarrenR.,
SampsonS. L.,
RichardsonM.,
van der SpuyG. D.,
LombardC. J.,
VictorT. C.,
van HeldenP.2000; Mapping of IS 6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity. Mol Microbiol 37:1405–1416[CrossRef]
WilsonM.,
De RisiJ.,
KristensenH. H.,
ImbodenP.,
RaneS.,
BrownP. O.1999; Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. Proc Natl Acad Sci U S A 96:12833–12838[CrossRef]