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Mutational and expression analysis of tbnat and its response to isoniazid

¹MRC Centre for Molecular and Cellular Biology and Department of Medical Biochemistry, University of Stellenbosch, Faculty of Health Sciences, PO Box 19063, Tygerberg, 7505, South Africa ²Department of Pharmacology, University of Oxford, Oxford, UK

Correspondence Paul D. van Helden pvh{at}sun.ac.za

Received 11 May 2005
Accepted 10 August 2005

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.