The impact of pyrazinamide on metabolism in Mycobacterium tuberculosis

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Project Summary Pyrazinamide (PZA) is a critical component of first-line tuberculosis (TB) therapy because it has dramatically reduced relapse rates and treatment duration. PZA is weakly active in vitro, but potently sterilizing in vivo, due to its outstanding activity against slow and non-replicating populations of Mycobacterium tuberculosis that are phenotypically tolerant to most other TB drugs. Understanding the basis for the in vivo sterilizing activity of PZA represents one of the most important unmet needs in TB drug discovery. While the mode of action of PZA remains under investigation, our knowledge of factors that govern susceptibility and resistance has advanced considerably in recent years. PZA is a pro-drug that must be converted to the active form of pyrazinoic acid (POA) by the M. tuberculosis amidase PncA, and loss-of-function mutations in pncA account for at least 70% of clinical resistance. Other genetic variations associated with resistance have been described, but most have not been evaluated in isogenic strains to determine their role in resistance, and have not been assessed for association with PZA resistance in animal models of infection. Importantly, PZA lacks antitubercular activity in athymic nude mice, indicating a key role for T cell-mediated immunity in efficacy. Consistent with these observations, our lab and others have shown that PZA is inactive against M. tuberculosis in resting macrophages, but contributes to bacterial killing in macrophages activated by interferon-gamma (IFN-ɣ). Host- dependent stressors that are associated with IFN-ɣ stimulation, such as exposure to low pH, nutrient limitation and reactive oxygen species, have been implicated as contributors to PZA action. Our recent studies have revealed a central role for specific M. tuberculosis stress responses in PZA conditional susceptibility. Through these studies, we have identified a network of functions that modulates PZA susceptibility when expression of the corresponding genes is altered. We have also identified specific host factors that contribute to PZA action in macrophages and in infected mice. Based on these findings, we have identified means to bolster drug action under conditions where PZA typically lacks activity. Through this proposal, we aim to characterize novel molecular mechanisms for PZA resistance, determine host factors that modulate PZA susceptibility, and assess opportunities in host- and microbe-targeted PZA potentiation. These studies will advance our understanding of mechanisms that govern PZA susceptibility and resistance of M. tuberculosis. As a substantial proportion of TB disease results from impaired T cell responses, it is of fundamental importance to understand how these responses relate to PZA efficacy and how we can use this information to optimize PZA action in the context of impaired immunity.