Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity

Abstract

Mycobacteria are known to acquire resistance to the antituberculous drug pyrazinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZase), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PZA against bacteria. Additional mechanisms of resistance to the drug are known to exist but have not been fully investigated. Among these is the non-uptake of the pro-drug, a possibility investigated in the present study. The uptake mechanism of PZA, a requisite step for the activation of the pro-drug, was studied in Mycobacterium tuberculosis. The incorporation of [14C]PZA by the bacilli was followed in both neutral and acidic environments since PZA activity is known to be optimal a t acidic pH. By using a protonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, arsenate and low temperature, it was shown that an ATP-dependent transport system is involved in the uptake of PZA. Whilst the structurally analogous compound nicotinamide inhibited the transport system of PZA, other structurally related compounds such as pyrazinoic acid, isoniazid and cytosine did not. Acidic conditions were also without effect. Based on diffusion experiments in liposomes, it was found that PZA diffuses rapidly through membrane bilayers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diffusion of the drug. This finding may explain why the cell wall mycolate hydrophobic layer does not represent the limiting step in the diffusion of PZA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40 O/ O less covalently linked mycolates. PZase activity, and PZA uptake and susceptibility in different mycobacterial species were compared. M. tuberculosis, a naturally PZA-susceptible species, was the only species that exhibited both PZase activity and PZA uptake; no such correlation was observed with the four naturally resistant species examined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacterium avium used, with PZA uptake comparable to that of M. tuberculosis. Mycobacterium bovis BCG and Mycobacterium kansasii exhibited neither a PZase activity nor PZA uptake. These data clearly demonstrate that one of the mechanisms of resistance to PZA resides in the failure of strains to take up the drug, indicating that susceptibility to PZA in mycobacteria requires both the presence of a functional PZase and a PZA transport system. No correlation was observed between the occurrence and cellular location of PZase and of nicotinamidase in the strains examined, suggesting that one or both amides can be hydrolysed by other mycobacterial amidases.

Characterization ofpncAMutations of Pyrazinamide-ResistantMycobacterium tuberculosisin Korea

“…This implies that other mechanisms are involved in PZA resistance. Recently, two alternative mechanisms have been proposed: active efflux of bactericidal pyrazinoic acid from the organism (28) and defects in PZA uptake of the organism (29,30). Defects in other genes, such as those required for PZase expression, might also be a source of PZA resistance.…”

Old and New TB Drugs: Mechanisms of Action and Resistance

“…The relatively high degree of diversity in pncA mutations among PZA-resistant clinical isolates has complicated the development of molecular assays for the rapid and economical detection of PZA resistance. A small percentage of isolates with high-level PZA resistance contain no mutations in pncA or its promoter, suggesting alternative mechanisms of resistance such as deficient uptake (Raynaud, Laneelle et al 1999), enhanced efflux, or altered pncA regulation.…”

“…PZA has been hypothesized to act against bacilli residing in acidified compartments of the lung that are present during the early inflammatory stages of infection , since the drug's sterilizing activity appears to be limited to the first 2 months of therapy (1986; 1986; 1991). PZA enters tubercle bacilli passively and via an ATP-dependent transport system (Raynaud, Laneelle et al 1999). Intracellular accumulation of the drug occurs because of an inefficient efflux system unique to M. tuberculosis.…”

Identification of Enzymatic Catalysis of PncA using 1H-NMR

“…The PncA is located in the cytoplasm of M. tuberculosis and is expressed constitutively. 8,9 Recent reports that mutations in the pncA gene encoding PncA lead to the loss of PncA activity, have implicated that the loss of PncA activity is highly related with PZA-resistance. [9][10][11] The studies on physicochemical properties and structure-activity relationship of M.…”