Affiliation:
1. MRC/SAIMR/WITS Molecular Mycobacteriology Research Unit, South African Institute for Medical Research, and Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, Johannesburg, South Africa
Abstract
ABSTRACT
A pyrazinamidase (PZase)-deficient
pncA
mutant of
Mycobacterium tuberculosis
, constructed by allelic exchange, was used to investigate the effects of heterologous amidase gene expression on the susceptibility of this organism to pyrazinamide (PZA) and related amides. The mutant was highly resistant to PZA (MIC, >2,000 μg/ml), in accordance with the well-established role of
pncA
in the PZA susceptibility of
M. tuberculosis
(A. Scorpio and Y. Zhang, Nat. Med. 2:662–667, 1996). Integration of the
pzaA
gene encoding the major PZase/nicotinamidase from
Mycobacterium smegmatis
(H. I. M. Boshoff and V. Mizrahi, J. Bacteriol. 180:5809–5814, 1998) or the
M. tuberculosis pncA
gene into the
pncA
mutant complemented its PZase/nicotinamidase defect. In both
pzaA
- and
pncA
-complemented mutant strains, the PZase activity was detected exclusively in the cytoplasm, suggesting an intracellular localization for PzaA and PncA. The
pzaA
-complemented strain was hypersensitive to PZA (MIC, ≤10 μg/ml) and nicotinamide (MIC, ≥20 μg/ml) and was also sensitive to benzamide (MIC, 20 μg/ml), unlike the wild-type and
pncA
-complemented mutant strains, which were highly resistant to this amide (MIC, >500 μg/ml). This finding was consistent with the observation that benzamide is hydrolyzed by PzaA but not by PncA. Overexpression of PzaA also conferred sensitivity to PZA, nicotinamide, and benzamide on
M. smegmatis
(MIC, 150 μg/ml in all cases) and rendered
Escherichia coli
hypersensitive for growth at low pH.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology