Control of Oxidative Stress and Intracellular Survival in Francisella tularensis Live Vaccine Strain (LVS) via Acyl-CoA Synthetase

Francisella tularensis is a highly infectious, Gram-negative intracellular bacterium and the causative agent of tularemia, a potentially fatal disease. Owing to its low infectious dose, ease of aerosolization, high virulence, lack of an effective vaccine, and potential use as a bioterrorism agent, F. tularensis is classified by the CDC as a Tier 1 Category A Select Agent. Despite its clinical importance, the mechanisms underlying F. tularensis virulence remain incompletely understood. In this study, we generated a partial Tn5 transposon insertion mutant library in the F. tularensis live vaccine strain (LVS) and identified a mutant disrupted in the FTL_0690 gene through screening under macrophage-like conditions. FTL_0690 encodes an acyl-CoA synthetase. Characterization of both a transposon-insertion mutant and a targeted deletion mutant ({Delta}FTL_0690) revealed critical roles for this enzyme in F. tularensis pathobiology. Loss of FTL_0690 increased sensitivity to oxidative stress and impaired intracellular growth within macrophages compared to wild-type F. tularensis LVS. Lipidomic profiling of the {Delta}FTL_0690 mutant revealed disruptions in fatty acid metabolism, membrane lipid remodeling, and redox homeostasis. Altered lipid-derived and membrane-associated metabolites indicated defective phospholipid incorporation and altered membrane composition, likely contributing to oxidative stress sensitivity and reduced intramacrophage survival. Collectively, these findings demonstrate that FTL_0690 which encodes long-chain acyl-CoA synthetase, contributes to lipid homeostasis, membrane integrity, and oxidative stress resistance of F. tularensis. ImportanceThis work addresses critical gaps in our understanding of Francisella tularensis virulence by identifying lipid metabolism as a central determinant of intracellular survival and stress resistance. By integrating transposon mutagenesis, targeted gene deletion, and lipidomic profiling, this study provides mechanistic insight into how metabolic remodeling supports pathogenesis. Our identification and characterization of FTL_0690 as a long-chain acyl-CoA synthetase essential for lipid homeostasis, membrane integrity, and oxidative stress resistance reveals a previously unappreciated link between fatty acid metabolism and intramacrophage survival of F. tularensis.