Tolfenpyrad displays Francisella -targeted antibiotic activity that requires an oxidative stress response regulator for sensitivity.

Francisella tularensis is a highly infectious Gram-negative bacterial pathogen capable of animal-to-human transmission. Due to its remarkable pathogenicity and potential for widespread public health impact, F. tularensis is classified as a high-priority pathogen by the Centers for Disease Control and Prevention. The historical development of Francisella as a bioweapon, coupled with the ease of engineering antibiotic-resistant strains, has spurred interest in the discovery of novel antibiotics. A library of drug-like compounds was screened for their ability to inhibit the growth of Francisella novicida , a biosafety level-2 surrogate for F. tularensis . Tolfenpyrad, a pesticide without previously identified antibacterial activity, was among the most potent inhibitors. Tolfenpyrad appears to uniquely target Francisella , as related species were sensitive, whereas bacteria in other genera were resistant. To identify Francisella pathways conferring sensitivity to tolfenpyrad, whole-genome sequencing of tolfenpyrad-resistant isolates was performed. Point mutations in two genes conferred resistance to osrR, a transcriptional regulator, and nuoM, a subunit of the electron transport chain. OsrR point mutants displayed attenuated growth in interferon gamma (IFN-γ)-stimulated macrophages but not in untreated macrophages, indicating that OsrR plays a role in resistance to IFN-γ-stimulated clearance of intracellular bacteria. OsrR point mutants are hypersensitive to reactive oxygen species, suggesting a mechanism by which they are rapidly cleared from IFN-γ-treated macrophages. This work identifies a novel Francisella -specific antibiotic activity and the metabolic pathways targeted. Moreover, we provide amino acid-level details of a Francisella protein that governs sensitivity to free radicals.IMPORTANCE Francisella species are highly pathogenic bacteria that pose a threat to global health security. These bacteria can be made resistant to antibiotics through facile methods, and we lack a safe and protective vaccine. Given their history of development as bioweapons, new treatment options must be developed to bolster public health preparedness. Here, we report that tolfenpyrad, a pesticide that is currently in use worldwide, effectively inhibits the growth of Francisella . This drug has an extensive history of use and a plethora of safety and toxicity data, making it a good candidate for development as an antibiotic. We identified mutations in Francisella novicida that confer resistance to tolfenpyrad and characterized a transcriptional regulator that is required for sensitivity to both tolfenpyrad and reactive oxygen species.