Cytosolic Fe-superoxide dismutase safeguards<i>Trypanosoma cruzi</i>from macrophage-derived superoxide radical

Abstract

<i>Trypanosoma cruzi</i>, the causative agent of Chagas disease (CD), contains exclusively Fe-dependent superoxide dismutases (Fe-SODs). During <i>T. cruzi</i> invasion to macrophages, superoxide radical (O₂^•-) is produced at the phagosomal compartment toward the internalized parasite via NOX-2 (gp91-<i>phox</i>) activation. In this work, <i>T. cruzi</i> cytosolic Fe-SODB overexpressers (pRIBOTEX-Fe-SODB) exhibited higher resistance to macrophage-dependent killing and enhanced intracellular proliferation compared with wild-type (WT) parasites. The higher infectivity of Fe-SODB overexpressers compared with WT parasites was lost in gp91-<i>phox</i>^-/- macrophages, underscoring the role of O₂^•- in parasite killing. Herein, we studied the entrance of O₂^•- and its protonated form, perhydroxyl radical [(HO₂^•); pK_a = 4.8], to <i>T. cruzi</i> at the phagosome compartment. At the acidic pH values of the phagosome lumen (pH 5.3 ± 0.1), high steady-state concentrations of O₂^•- and HO₂^• were estimated (∼28 and 8 µM, respectively). Phagosomal acidification was crucial for O₂^•- permeation, because inhibition of the macrophage H⁺-ATPase proton pump significantly decreased O₂^•- detection in the internalized parasite. Importantly, O₂^•- detection, aconitase inactivation, and peroxynitrite generation were lower in Fe-SODB than in WT parasites exposed to external fluxes of O₂^•- or during macrophage infections. Other mechanisms of O₂^•- entrance participate at neutral pH values, because the anion channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid decreased O₂^•- detection. Finally, parasitemia and tissue parasite burden in mice were higher in Fe-SODB-overexpressing parasites, supporting the role of the cytosolic O₂^•--catabolizing enzyme as a virulence factor for CD.