Trypanothione reductase overexpression increases the infectivity of Leishmania tarentolae through improved redox homeostasis and increased resistance to oxidative stress.

Leishmaniasis is a neglected tropical disease that imposes a significant global health burden. To establish persistent infections, parasites must overcome oxidative stress generated by host immune responses, a process for which the parasite-specific enzyme trypanothione reductase (TR) is critical. While TR has been implicated in infectivity, its precise role in governing infectivity remains incompletely understood. This study aimed to investigate the hypothesis that TR activity is a key determinant of parasite infectivity. We first compared TR activity between pathogenic Leishmania infantum and nonpathogenic L. tarentolae and found that L. infantum exhibits 5.7-fold higher TR activity than L. tarentolae. Structural analysis revealed significant amino acid substitutions (Asp116Glu and Lys240Gln) in L. tarentolae TR that likely account for the observed reduction in TR activity. To functionally validate the role of TR, we generated L. tarentolae lines overexpressing TR. Compared with wild-type L. tarentolae, these mutants exhibit 6-fold higher enzyme activity (p < 0.0001) and significantly increased resistance to hydrogen peroxide (IC50: 515 µM vs. 425 µM in the wild type; p < 0.0001). Consequently, TR-overexpressing parasites show a dramatic increase in infectivity in murine peritoneal macrophages (8-fold at 24 hours; 78-fold at 72 hours). Most notably, in vivo infection experiments in BALB/c mice (n=5 per group) revealed that compared with wild-type parasites, TR-overexpressing parasites result in a 3.3-fold higher liver parasite load. These findings establish that TR activity is an important infectivity-associated factor that contributes substantially to parasite infectivity by increasing antioxidant capacity. Our results strongly support TR as a high-priority drug target, suggesting that its inhibition represents a promising strategy for developing novel drugs against leishmaniasis and other kinetoplastid infections.