Selective inhibition of respiratory complex I reveals a bioenergetic vulnerability in Francisella

F. tularensis is a highly infectious Gram-negative bacterial pathogen that causes tularemia, a re-emerging zoonosis of public health concern. Here we identify respiratory complex I as a selective vulnerability in Francisella and define the mechanism of action of a pyrazole compound, tolfenpyrad, with species-specific antibacterial activity. Using F. novicida as a surrogate model, we demonstrated that tolfenpyrad selectively inhibits growth with no measurable effect on E. coli or P. aeruginosa . Tolfenpyrad rapidly suppressed oxygen consumption, depleted ATP, collapsed proton motive force, and induced reactive oxygen species, indicating disruption of bacterial metabolism. Biochemical assays demonstrated selective inhibition of NADH-dependent respiration and membrane-associated NADH oxidation, whereas succinate-driven respiration was unaffected. Moreover, the alternative NADH dehydrogenase ( ndh ) was not required for tolfenpyrad activity. Structural docking identified a potential tolfenpyrad-binding pocket within the membrane subunit NuoM. These findings reveal species-specific inhibition of Francisella complex I and establish respiratory metabolism as a promising antimicrobial target in these bacteria.