Role of TbTim17 in mitochondrial stress tolerance in Trypanosoma brucei

Mitochondrial protein translocases Tim17 and Tim23 play important roles in stress-response pathways via activating the transcription factors, ATFS-1, to maintain organellar homeostasis. Trypanosoma brucei, a divergent eukaryote and the infectious agent for African trypanosomiasis, lacks ATFs but possesses TbTim17, an essential component of the TIM complex in mitochondria. However, it has not been investigated whether TbTim17 plays a role in the mitochondrial stress response. Here, we show that depletion of TbTim17 increased T. brucei tolerance to paraquat, increasing the EC50 by 3- to 4-fold compared with the wild type. Subsequent analysis revealed that increased levels of mitochondrial reactive oxygen species resulting from TbTim17 knockdown upregulate mitochondrial superoxide dismutase, thereby preadapting cells to resist paraquat-induced oxidative stress. This is supported by the finding that treating cells with N-acetyl cysteine during TbTim17 RNAi induction reduced the EC50 of paraquat to wild-type levels. TbTim17 knockdown also increased tolerance of T. brucei to heat stress. Either heat or oxidative stress did not increase mitochondrial heat shock protein 70 or Bip levels in the ER in T. brucei; instead, they moderately increased TbTim17 levels, which can replenish mitochondrial proteomes. Furthermore, TbTim17 knockdown caused a significant reduction in SL RNA and a 2- to 5-fold increase in tSNAP42 transcript levels, suggesting that mitochondrial stress is linked to the ER stress response pathway in T. brucei. Together, these results show that the mitochondrial stress response is primarily mediated by antioxidant defense mechanisms and that TbTim17 plays a protective role in mitochondria under stress in T. brucei. ImportanceTrypanosoma brucei, a parasitic protozoan, is the infectious agent of a deadly disease in humans and livestock known as African trypanosomiasis. TbTim17 is the major component of the TbTIM complex that imports hundreds of nuclear-encoded proteins into the mitochondrial matrix and inner membrane. Here, we show that depletion of TbTim17 induces oxidative stress, which upregulates mitochondrial antioxidant defense mechanisms to mitigate this stress. Subsequently, the SLS response is induced to eliminate the defective parasite from the population. In the wild-type parasite, TbTim17 levels are increased in mitochondria under oxidative and heat stresses, likely to replenish damaged mitochondrial proteomes. This is unlike in other eukaryotes, where oxidative stress degrades Tim17 to induce mitochondrial stress response. Understanding the mitochondrial stress response in T. brucei and the role of mitochondrial protein translocases in this process is critical for elucidating the mechanisms of adaptation to environmental stresses and drug resistance in this parasite.