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.

非洲锥虫可表达三种几乎完全相同的不含硒谷胱甘肽过氧化物酶（non-selenium glutathione peroxidase, Px），此类酶优先解毒脂质源性过氧化物。既往研究显示，缺失线粒体Px III的血液阶段布氏锥虫（Trypanosoma brucei）仅表现出轻微且短暂的增殖缺陷；而缺失胞质Px I与Px II的寄生虫则会发生极快速的脂质过氧化与细胞裂解。该表型可通过在培养基中添加α-生育酚衍生物Trolox完全挽救。然而，这种快速细胞死亡背后的机制始终不明。本研究证实溶酶体（lysosome）是细胞损伤的起源。当在添加Trolox的条件下用Alexa Fluor偶联葡聚糖（Alexa Fluor-conjugated dextran）或LysoTracker染料标记px I-II敲除寄生虫时，可观察到清晰的溶酶体染色信号。但当移除抗氧化剂后，该信号会逐渐扩散至整个细胞胞体并最终完全消失。布氏锥虫通过内吞宿主转铁蛋白获取铁离子：在培养基中补充铁离子或转铁蛋白会诱导px I-II敲除细胞的裂解，而铁螯合剂去铁胺（deferoxamine）与脱铁转铁蛋白（apo-transferrin）则会抑制该裂解过程。使用MitoTracker染料与溶酶体标记蛋白p67抗体进行免疫荧光显微镜观察发现，溶酶体的解体早于线粒体损伤。体内实验进一步验证了线粒体过氧化物酶的可忽略作用：感染px III敲除细胞的小鼠，其疾病进程仅较野生型寄生虫感染组略微延迟。本研究数据表明，在血液阶段的非洲锥虫中，溶酶体而非线粒体是氧化损伤的主要位点，胞质锥硫酮/锥氧还蛋白依赖性过氧化物酶可保护溶酶体免受铁诱导的膜脂质过氧化。该过程似乎与布氏锥虫感染阶段的高内吞速率以及独特的铁获取机制密切相关。在昆虫阶段的前循环型（procyclic insect form）寄生虫中敲除胞质px I-II后，细胞可在不含Trolox的培养基中正常存活。