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