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.

Department of Cell and Developmental Biology Biocenter University Wuerzburg Wuerzburg Germany

Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

Institute of Molecular Biology Mainz Germany

Institute of Parasitology Biology Centre Czech Academy of Sciences Ceske Budejovice Czech Republic

Welcome Centre for Integrative Parasitology Institute of Infection Immunity and Inflammation Glasgow Polyomics College of Medical Veterinary and Life Sciences University of Glasgow Glasgow United Kingdom

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Redesigned and reversed: architectural and functional oddities of the trypanosomal ATP synthase

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei