The cytosolic or the mitochondrial glutathione peroxidase-type tryparedoxin peroxidase is sufficient to protect procyclic Trypanosoma brucei from iron-mediated mitochondrial damage and lysis.

African trypanosomes express three virtually identical glutathione peroxidase (Px)-type enzymes that occur in the cytosol (Px I and II) and mitochondrion (Px III) and detoxify fatty acid-derived hydroperoxides. Selective deletion of the genes revealed that procyclic Trypanosoma brucei lacking either the cytosolic or mitochondrial enzyme proliferate nearly as wild-type parasites, whereas the knockout of the complete genomic locus is lethal. Flow cytometry and immunofluorescence analyses revealed that the Px I-III-deficient parasites lose their mitochondrial membrane potential, which is followed by a loss of the lysosomal signal but not the glycosomal one. Mitochondrial damage and cell lysis are prevented by Trolox, ubiquinone derivatives and the iron chelator deferoxamine, whereas starch-deferoxamine is inefficient. In glucose-rich medium, cell death is attenuated suggesting that oxidants generated by the respiratory chain contribute to the lethal phenotype. Thus, the Px-type peroxidases protect procyclic cells from an iron-mediated oxidative membrane damage that originates at the mitochondrion. This contrasts with the situation in bloodstream cells, where the lysosome is the primarily affected organelle. Strikingly, either the cytosolic or the mitochondrial form of the peroxidases is required and sufficient to protect the mitochondrion and prevent cell lysis.

Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px)-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective stage of T. brucei. The respective knockout of the cytosolic px I-II in the procyclic insect form resulted in cells that were fully viable in Trolox-free medium.