Project summary/Abstract: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) and is the leading cause of death due to a single bacterium. In 2020, around 10 million people developed TB worldwide and approximately 1.5 million people died from TB. TB is curable but the treatment is toxic and long (minimum of 6 month). In the case of drug resistant TB, the treatment lasts even longer (9 to 24 months) and the chances of patient survival are reduced. Among the 4 first-line anti-TB drugs used to treat TB, pyrazinamide (PZA) is notorious for its poor activity against Mtb in vitro. PZA was originally identified in a screen performed in Mtb-infected animals which explains why the poor activity of PZA in vitro did not stop its discovery as a major anti-TB drug. The main consequence of the poor activity of PZA in vitro is the lack of understanding of PZA mode of action which results in a reduced capacity to fight against PZA-resistant TB. We recently established an in vitro model in which PZA displays a high bactericidal activity against Mtb. In this application, we will validate the involvement of the genes/pathways identified in a genetic screen to alter PZA susceptibility by constructing selected mutants and measuring their susceptibility to PZA. Moreover, we will also isolate spontaneous PZA-resistant clones in order to identify bona fide PZA targets and measure the frequency of resistance to PZA in our in vitro model. The PZA susceptibility profile of the mutants generated will then be validated in a macrophage model of TB infection. In addition to the identification of genetic determinants for PZA susceptibility, we will characterize how PZA impacts Mtb metabolism. Using our in vitro model, we will analyze the metabolome of Mtb using metabolomics and we will measure the impact of PZA on Mtb respiratory chain functions. Mutants with altered susceptibility to PZA will be used in addition to wild-type Mtb to elucidate the mechanism of action of PZA. We anticipate that this project will advance our knowledge on the mechanism of action of PZA and will help the fight against PZA-resistant TB. We believe this study will also pave the way for the use of in vitro conditions more closely related to the infection settings in order to improve the discovery and the study of drugs to fight against TB and other infectious diseases.