Chemical genetic interactions elucidate pathways controlling tuberculosis antibiotic efficacy during infection

Successful tuberculosis therapy requires treatment with an unwieldy multidrug combination for several months. Thus, there is a growing need to identify novel genetic vulnerabilities that can be leveraged to develop new, more effective antitubercular drugs. Consequently, recent efforts to optimize tuberculosis (TB) therapy have exploited Mycobacterium tuberculosis (Mtb) chemical genetics to identify pathways influencing antibiotic efficacy, novel mechanisms of antibiotic action, and new targets for TB drug discovery. However, the influence of the complex host environment on these interactions remains largely unknown, leaving the therapeutic potential of the identified targets unclear. In this study, we leveraged a library of conditional mutants targeting 467 essential Mtb genes to characterize the chemical–genetic interactions (CGIs) with TB drugs directly in the mouse infection model. We found that these in vivo CGIs differ significantly from those identified in vitro. Both drug-specific and drug-agnostic effects were identified, and many were preserved during treatment with a multidrug combination, suggesting numerous strategies for enhancing therapy. This work also elucidated the complex effects of pyrazinamide (PZA), a drug that relies on aspects of the infection environment for efficacy. Specifically, our work supports the importance of coenzyme A synthesis- inhibition during infection, as well as the antagonistic effect of iron limitation on PZA activity. In addition, we found that inhibition of thiamine and purine synthesis increases PZA efficacy, suggesting additional therapeutically exploitable metabolic dependencies. Our findings present a map of the unique in vivo CGIs, characterizing the mechanism of PZA activity in vivo and identifying potential targets for TB drug development.