Tolfenpyrad Modulates Antibiotic Activity on Francisella novicida and Synergizes with Electron Transport Chain Inhibitors

Francisella tularensis is a highly virulent intracellular pathogen for which treatment options remain limited and vulnerable to resistance development. Tolfenpyrad, a pesticide with selective antibacterial activity against Francisella species, has emerged as a promising compound, yet its mechanism of action remains poorly defined. Here, we investigated how tolfenpyrad modulates the activity of established antibiotics and probed its effects on bacterial energy metabolism in Francisella . In this study, we used combinatorial drug experiments to identify both synergistic and antagonistic drug–drug interactions. Tolfenpyrad synergized with polymyxin B and azithromycin to inhibit Francisella novicida in vitro, and this synergistic activity was also observed with azithromycin in an intramacrophage infection model, resulting in enhanced bacterial clearance. In contrast, tolfenpyrad antagonized aminoglycoside and tetracycline-class antibiotics, restoring bacterial survival under otherwise inhibitory conditions. Consistent with this antagonism, tolfenpyrad attenuated uptake of tetracycline and doxycycline, suggesting that it may disrupt proton motive force–dependent transport. Supporting this model, tolfenpyrad synergized with multiple electron transport chain inhibitors in F. novicida and in F. tularensis, and reduced ATP levels in F. novicida . Collectively, these data indicate that tolfenpyrad disrupts bacterial energy metabolism in Francisella, likely impairing oxidative phosphorylation and proton motive force generation. These findings define a mechanistic framework for tolfenpyrad’s antibacterial activity and highlight its potential as an inhibitor of Francisella bioenergetics and a potential partner in combinatorial antibiotic therapies.