C. Raynaud et al., Mechanisms of pyrazinamide resistance in mycobacteria: importance of lack of uptake in addition to lack of pyrazinamidase activity, MICROBIO-UK, 145, 1999, pp. 1359-1367
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.