Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite’s metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro–induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro–induced differentiation. Our results provide insights into the mechanisms of the parasite’s mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.

线粒体代谢重塑是布鲁氏锥虫的标志昆虫阶段利用了具有成本效益的氧化磷酸化（OxPhos）来生成ATP，而血液细胞则切换到有氧糖酵解，从而实现了双基因生命周期。由于难以从采采蝇蝇载体中获得足够的寄生虫，因此该载体中寄生虫的代谢重新连接的动力学仍然不清楚。在这里，我们利用体外诱导的分化来追踪RNA，蛋白质和代谢产物水平的变化。这种多组学和基于细胞的谱分析表明，电子流立即从细胞色素介导的途径重定向到另一种氧化酶（AOX），脯氨酸消耗增加，复合物II的活性升高，某些三羧酸（TCA）循环酶，导致线粒体膜超极化并增加活性氧（ROS）水平。有趣的是，这些ROS分子似乎是驱动发育进程的信号分子，因为过氧化氢酶（一种ROS清除剂）的异位表达阻止了体外诱导的分化。我们的结果提供了对寄生虫的线粒体重新布线机制的见解，并加强了线粒体通过释放ROS来驱动细胞分化而充当信号细胞器的新概念。