Systematic analysis of pyrazinamide-resistant spontaneous mutants and clinical isolates of Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZA(r)) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10(-5) bacilli in vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZA(r) phenotype correlated with mutations in the pncA gene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZA(r) mutants did not have a pncA mutation, strongly indicating the presence of at least one other mechanism(s) of PZA(r).

Fig 1

3D structure of pyrazinamidase showing that mutations cover the entire protein structure. The ribbon is shown in cream. The α-carbon atom of each mutated residue is shown as a colored ball. Green balls indicate neutral or stabilizing mutations (M1, A46, Y64, H82, T87, Y103, L116, T135, M175, and L182); the green residues A46, H82, and Y103 are in the vicinity of the active site, which could correlate with the loss of enzyme activity. Blue balls indicate destabilizing residues (A3, D12, F13, C14, G24, L27, I31, L35, H43, V44, S59, D63, S67, W68, T76, L85, S104, T135, D136, R140, Q141, A143, A146, R154, V155, L159, A171, and L172). Red balls indicate residues located in the active site pocket or involved in chelating the Fe atom (orange) (D8, V9, Q10, D49, H51, P54, H57, W68, H71, C72, G132, A134, C138, V139, and T142). Note that 4 α-carbon atoms (residues L35, T87, G132, and D136) are not visible, as they are behind the ribbon (on the bottom), and therefore they are not labeled in the figure. The figure was produced by successively using the MolScript (26) and Raster3D (37) programs.