224 - Cytosolic Fe-Superoxide Dismutase Protects Trypanosoma Cruzi from Host-Derived Superoxide and Increases Pathogen Virulence in Vivo

Abstract

Trypanosoma cruzi, the causative agent of Chagas Disease, contains Fe-dependent superoxide dismutases (Fe-SODs). After invasion, parasites have to deal with large amounts of superoxide radical (O2●–) produced during macrophage phagocytosis (NADPH-oxidase activation) and, in cytokine-activated macrophages (iNOS induction), with nitric oxide (●NO) and peroxynitrite. In this work, we studied the relevance of T. cruzi cytosolic Fe-SOD (Fe-SODB) during infection in vitro and in vivo. Fe-SODB-overexpressing parasites (TcFeSODB) were generated using the plasmid pRIBOTEX and expression evaluated by western-blot, immunocytochemistry and activity measurements. TcFeSODB parasites had less intra-parasite peroxynitrite generation than wt when exposed to O2●–- and ●NO fluxes, as evaluated by the oxidation of fluorescein boronate. They were also more resistant to macrophage killing (24hs after infection to J774A.1 or primary macrophages). These enhanced resistance, provided by high Fe-SOD levels in T. cruzi, was lost in NADPH-oxidase knockout macrophages (from gp91-/- mice). In the phagosome (pH ≈ 5-6), O2●–could be protonated (HO2●, pKa = 4,88) and diffuse towards parasite cytosol. This was confirmed by exposing the parasites to external fluxes of O2●– (generated by the system xanthine-oxidase/xanthine) at acidic and basic pHs (6 and 8 respectively). Intra-parasite 2-hydroxyethidium content (O2●–-specific product of dihydroethidium oxidation) was larger at pH=6 than pH=8. Additional entry mechanisms also participate, as the anion channel inhibitor NPPB, but not DIDS, decreased 2-hydroxyethidium detection. Inhibition of macrophage’s v-ATPase proton pump and prevention of phagosome acidification also decreases 2-hydroxyethidium detection in trypomastigotes 2h after infection. These results clearly show that O2●–can reach the parasite cytosol during phagocytosis. Finally, parasitemia in C57BL/6 infected mice (n=5) was larger (40%) for TcFeSODB than in wt parasites, demonstrating that Fe-SODB acts as a virulence factor in the mouse model of Chagas Disease.