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

Mycobacteria are known to acquire resistance to the antituberculous drug py razinamide (PZA) through mutations in the gene encoding pyrazinamidase (PZa se), an enzyme that converts PZA into pyrazinoic acid, the presumed active form of PEA against bacteria. Additional mechanisms of resistance to the dr ug 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 s tudy. The uptake mechanism of PEA, a requisite step for the activation of t he pro-drug, was studied in Mycobacterium tuberculosis. The incorporation o f [C-14]PZA by the bacilli was followed in both neutral and acidic environm ents since PEA activity is known to be optimal at acidic ph. By using a pro tonophore (carbonyl cyanide m-chlorophenylhydrazone; CCCP), valinomycin, ar senate and low temperature, it was shown that an ATP-dependent transport sy stem is involved in the uptake of PZA. Whilst the structurally analogous co mpound nicotinamide inhibited the transport system of PZA, other structural ly related compounds such as pyrazinoic acid, isoniazid and cytosine did no t. Acidic conditions were also without effect. Based on diffusion experimen ts in liposomes, it was found that PEA diffuses rapidly through membrane bi layers, faster than glycerol, whilst the presence of OmpATb, the porin-like protein of M. tuberculosis, in proteoliposomes slightly increased the diff usion of the drug. This finding may explain why the cell wall mycolate hydr ophobic layer does not represent the limiting step in the diffusion of PEA, as judged from comparative experiments using a M. tuberculosis strain and its isogenic mutant elaborating 40% less covarently linked mycolates. PZase activity, and PEA uptake and susceptibility in different mycobacterial spe cies were compared. M. tuberculosis, a naturally PEA-susceptible species, w as the only species that exhibited both PZase activity and PZA uptake; no s uch correlation was observed with the four naturally resistant species exam ined. Mycobacterium smegmatis possessed a functional PZase but did not take up PZA; the reverse was true for the PZase-negative strain of Mycobacteriu m avium used, with PZA uptake comparable to that of M. tuberculosis. Mycoba cterium bovis BCC and Mycobacterium kansasii exhibited neither a PZase acti vity nor PZA uptake. These data clearly demonstrate that one of the mechani sms of resistance to PEA resides in the failure of strains to take up the d rug, indicating that susceptibility to PZA in mycobacteria requires both th e 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.