Abstract
Francisella tularensis is an important human pathogen responsible for causing tularemia in the Northern Hemisphere. Francisella has been developed as a biological weapon in the past due to its extremely high virulence. F. tularensis is a gram-negative, intracellular pathogen that primarily infects macrophages. F. tularensis encodes a repertoire of antioxidant enzymes to counteract the reactive oxygen and nitrogen species (ROS/RNS) produced by macrophages in response to infection. Among these, the thioredoxin system is critical for maintaining cellular redox homeostasis by regulating the balance between oxidation and reduction within bacterial cells. This system includes thioredoxins, thioredoxin reductase, and NADPH. Despite its potential importance, the thioredoxin system of F. tularensis remains understudied. F. tularensis live vaccine strain (LVS) possesses two thioredoxin genes, trxA1 (FTL_0611) and trxA2 (FTL_1224), and a single thioredoxin reductase gene, trxB (FTL_1571). In this study, we characterized the role of trxB of F. tularensis LVS in oxidative stress resistance. Our findings demonstrate that trxB is essential for oxidative stress resistance in F. tularensis and that its loss increases susceptibility to several antibiotics. However, unlike other bacterial species, TrxB in F. tularensis is not a functional target of the gold-containing antimicrobial agent auranofin. We also show that OxyR, the master regulator of oxidative stress responses, directly controls trxB expression under oxidative stress conditions. Furthermore, TrxB contributes to intramacrophage survival by enabling the bacterium to withstand ROS-induced oxidative stress. Collectively, this study highlights a critical, previously uncharacterized antioxidant defense mechanism in F. tularensis and its importance in oxidative stress resistance and intramacrophage survival. IMPORTANCE: This study elucidates the function of the trxB gene, which encodes a thioredoxin reductase, in overcoming oxidative stress by Francisella tularensis. Loss of the trxB gene results in enhanced susceptibility to oxidants, diminished intracellular survival, and antibiotic resistance. Unlike other bacterial species, F. tularensis TrxB is not a functional target of auranofin, a gold-containing antimicrobial compound, suggesting divergence in thioredoxin system interactions. Furthermore, transcriptional regulation of trxB by OxyR in response to oxidative stress highlights an adaptive control mechanism essential to resist oxidative stress. These findings provide a mechanistic understanding of F. tularensis antioxidant defenses and their role in intramacrophage survival.