Mutually Exclusive Genotypes for Pyrazinamide and 5-Chloropyrazinamide Resistance Reveal a Potential Resistance-Proofing Strategy

The pyrazinamide (PZA) analog 5-chloropyrazinamide (5-Cl PZA) is active against mycobacterial species, including PZA-resistant strains of Mycobacterium tuberculosis . In M. smegmatis , overexpression of the type 1 fatty acid synthase (FAS I) confers resistance to 5-Cl PZA, a potent FAS I inhibitor. Since M. tuberculosis and M. bovis cannot tolerate FAS I overexpression, 5-Cl PZA resistance mutations have yet to be described for tubercle bacilli. In an attempt to identify other factors that govern the activity of 5-Cl PZA, we selected for 5-Cl PZA-resistant isolates from a library of transposon-mutagenized M. smegmatis isolates. Here, we report that increased expression of the M. smegmatis pyrazinamidase PzaA confers resistance to 5-Cl PZA and susceptibility to PZA in M. smegmatis , M. tuberculosis , and M. bovis . In contrast, while ectopic overexpression of the M. tuberculosis pyrazinamidase PncA increases PZA susceptibility, this amidase does not mediate resistance to 5-Cl PZA. We conclude that PncA-independent turnover of 5-Cl PZA represents a potential mechanism of resistance to this compound for M. tuberculosis , which will likely translate into enhanced PZA susceptibility. Thus, countersusceptibility can be manipulated as a resistance-proofing strategy for PZA-based compounds when these agents are used simultaneously.