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

Abstract

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.

Pantothenate and Pantetheine Antagonize the Antitubercular Activity of Pyrazinamide

“…Once in the cytoplasm, the proton dissociates from POA, and the cycle continues until the cytoplasmic pH equilibrates with that of the extracellular environment. Inconsistent with this model, the acid dependence of PZA activity can be abolished by incubation under alkaline pH (5), by overexpression of pncA (19), or by preventing POA from cycling in and out of the cell by the addition of efflux pump inhibitors (4). Moreover, it has been demonstrated that the primary in vivo niches for M. tuberculosis, phagocyte lysosomes and tubercular granulomas, do not acidify when live bacilli are present (20,21).…”

“…The first of these models comes from studies involving the PZA structural analog 5-chloropyrazinamide (5-Cl PZA), which was found to be a potent inhibitor of the mycobacterial fatty acid synthase I (FAS-I) (16)(17)(18). While M. tuberculosis FAS-I activity was found to also be compromised by PZA treatment (16,18,19), in situ evidence for this inhibition has been confounded by the inability to overexpress FAS-I in M. tuberculosis (16). The model has also been challenged by a report in which inhibition of purified FAS-I by POA was not observed, and FAS-I inhibition in whole cells was suggested to be a downstream consequence of POA-mediated organic acid stress (17).…”

Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

“…On the basis of these and other findings, a number of models have been proposed to explain the antimycobacterial action of PZA and POA, including direct inhibition of fatty acid synthase I (10), disruption of the membrane potential and cytoplasmic pH homeostasis via a protonophore activity (11), inhibition of transtranslation (12), and inhibition of the aspartate decarboxylase PanD, involved in pantothenate and coenzyme A (CoA) biosynthesis (13). While the bases for these models provide clues toward understanding PZA and POA susceptibility, fundamental aspects of these models have been challenged by recent reports (7,(14)(15)(16)(17)(18)(19). Consistent with a role for PanD in PZA action, it has recently been demonstrated that exogenously supplied intermediates of the CoA biosynthetic pathway can antagonize PZA activity, point mutations in panD are associated with in vitro PZA resistance, and PZA treatment leads to the depletion of intrabacterial levels of pantothenate and CoA (13,15,20).…”

Uncoupling Environmental pH and Intrabacterial Acidification from Pyrazinamide Susceptibility in Mycobacterium tuberculosis

“…Recent reports have revealed that the acid dependence of PZA action can be diminished through coincubation with efflux pump inhibitors (18,27) or by overexpression of PncA (28). Nonetheless, proton shuttling resulting in cytoplasmic acidification remains a plausible but untested model for PZA action.…”