Investigating Conditions That Modulate Pyrazinamide Susceptibility In Mycobacterium Tuberculosis

ABSTRACT Tuberculosis (TB) remains one of the major life threatening infectious diseases of public health concern globally. Its treatment is long and complex, requiring a cocktail of four drugs administered for six months. Pyrazinamide (PZA) is a cornerstone drug in the treatment regimen for tuberculosis that has contributed to reducing the treatment time from nine to six months. PZA is a pro-drug that is converted to its active form, pyrazinoic acid (POA) by the bacterial enzyme nicotinamidase. Unlike the other TB drugs, mechanistic basis for susceptibility and resistance to PZA are poorly understood. Recent studies have shown that PZA acts by depletion of thiol active coenzyme A (CoA) levels in M. tuberculosis. Using M. bovis BCG as a model organism, this study sought out to identify ways by which POA action can be potentiated, as well as identify novel POA resistance mechanisms. We also, tried to understand the mechanism by which host associated stresses potentiate PZA action. Himar1 mariner-based transposon mutagenesis was conducted to identify novel POA resistance conferring mutations in BCG. Using checkerboard assays, we tested the hypothesis that oxidative stress potentiates POA action by oxidizing thiol active CoA thereby reducing its abundance. We also overexpressed phosphoenolpyruvate carboxykinase (pckA), a key player in CoA metabolism, to ascertain if this can enhance susceptibility to POA. From the transposon screen, it was identified that phage infection potentiates POA action by reducing the minimum inhibitory concentration of POA from approximately 2500 µg/ml to 75 µg/ml, possibly through induction of cell envelope stress. In addition, 62 POA resistant transposon mutants were isolated and 4 novel POA conferring mutations were identified. Synergy was observed between PZA and H2O2 resulting in enhanced susceptibility. POA conversion was also shown to be required for iii this observed synergy with H2O2. Overexpression of pckA led to a 2-fold increased susceptibility of BCG to POA. We can conclude from this study that, host derived oxidative stress experienced by M. tuberculosis in macrophages, is potentially associated with POA action, and importantly, alterations in central carbon metabolism that modulate CoA abundance results in modulation of POA action.