Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is a first-line drug for short-course tuberculosis therapy. Resistance to PZA is usually accompanied by loss of pyrazinamidase (PZase) activity in Mycobacterium tuberculosis. PZase converts PZA to bactericidal pyrazinoic acid, and the loss of PZase activity is associated with PZA resistance. The gene (pncA) encoding the M. tuberculosis PZase has recently been sequenced, and mutations in pncA were previously found in a small number of PZA-resistant M. tuberculosis strains. To further understand the genetic basis of PZA resistance and determine the frequency of PZA-resistant strains having pncA mutations, we analyzed a panel of PZA-resistant clinical isolates and mutants made in vitro. Thirty-three of 38 PZA-resistant clinical isolates had pncA mutations. Among the five strains that did not contain pncA mutations, four were found to be falsely resistant and one was found to be borderline resistant to PZA. The 33 PZA-resistant clinical isolates and 8 mutants made in vitro contained various mutations, including nucleotide substitutions, insertions, or deletions in the pncA gene. The identified mutations were dispersed along the pncA gene, but some degree of clustering of mutations was found at the following regions: Gly132-Thr142, Pro69-Leu85, and Ile5-Asp12. PCR-single-strand conformation polymorphism (SSCP) analysis was shown to be useful for the rapid detection of pncA mutations in the PZA-resistant strains. We conclude that a mutation in the pncA gene is a major mechanism of PZA resistance and that direct sequencing by PCR or SSCP analysis should help to rapidly identify PZA-resistant M. tuberculosis strains.

Current perspectives in drug discovery against tuberculosis from natural products

“…Pyrazinamide (PZA), an analog of nicotinamide (Fig. 2a), is a prodrug that requires conversion by MTB pyrazinamidase (coded by the pncA gene) to pyrazinoic acid [16]. RpsA has recently been shown to be a pyrazinamide cellular target [17], and its over-expression (wild-type RpsA) has been implicated in PZA resistance in MTB.…”

Characterization of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis isolates from Korea and analysis of the correlation between the mutations and pyrazinamidase activity

“…Mutations of pncA lead to the loss of PncA enzyme activity and constitute the mechanism of PZA resistance in Mycobacterium tuberculosis (Morlock et al 2000). The mutations causing PZA resistance have been well characterized worldwide (Hirano et al 1997;Scorpio et al 1997;Sreevatsan et al 1997;Lemaitre et al 1999;Marttila et al 1999;Lee et al 2001;Huang et al 2003;Portugal et al 2004; Barco et al 2006;Zhang et al 2009;Zimic et al 2010). The reported pncA mutations are largely missense mutations, leading to codon changes and subsequent amino acid substitutions, and in some cases, nucleotide insertions or deletions and nonsense mutations in the pncA structural gene or in the putative promoter region of pncA (Sreevatsan et al 1997).…”

Pyrazinamide inhibits the eukaryotic-like fatty acid synthetase I (FASI) of Mycobacterium tuberculosis

“…3). Pyrazinamidase is encoded by the pncA gene and PZA resistance in strains of M. tuberculosis and M. bovis (which is inherently resistant to PZA) is due to mutations in pncA that abolishes the amidase activity 8,9 . M. tuberculosis is the only mycobacterial species that is susceptible to PZA, pyrazinoic acid (POA) and NAM 10 .…”

“…Attempts to isolate POA resistant mutants of M. tuberculosis have been unsuccessful 9 . However, since different PZA analogs are active against M. tuberculosis and other strains of mycobacteria 13 , it is likely that these analogs affect the same target as PZA because essential functions are not redundant.…”