Abstract
The mitochondrial FoF1-ATP synthase is a reversible nanomachine that normally produces ATP via oxidative phosphorylation but under stress conditions it can reverse to maintain the mitochondrial membrane potential at the expense of ATP, a process regulated by the conserved inhibitory factor 1 (IF1). We show that ATP synthase reversal also occurs during in vitro-induced differentiation of the unicellular parasite Trypanosoma brucei, partially mirroring events in the tsetse fly. Differentiation of insect forms is marked by increased expression of alternative oxidase and reduced levels of trypanosomal IF1 (TbIF1), changes that may promote ATP synthase reversal. Parasites lacking TbIF1 efficiently progressed to the mammalian-infective form, coinciding with increased ATP synthase reversal, a higher ADP/ATP ratio, elevated phosphorylation of AMP-activated protein kinase (AMPK), enhanced proline-supported respiration, and increased mitochondrial and cellular reactive oxygen species (ROS). In contrast, inducible TbIF1 overexpression diminished these hallmarks and locked parasites in the initial insect stage. Our findings reveal that TbIF1 downregulation enables life cycle progression and underscore a regulatory role for the ATP synthase–IF1 axis.
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Data availability
All data supporting the findings of this study, including the uncropped and unprocessed Western blot images (Supplementary Fig. 4), are available within the paper. The source data for all charts/graphs, description of T. brucei strains, and list of used oligonucleotides can be found in Supplementary Data 1. All other data are available from the corresponding author on reasonable request.
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Acknowledgements
We would like to thank Martina Slapničková for excellent technical support and Prof. Christos Chinopoulos (Semmelweis University, Budapest) for stimulating discussions. We would also like to express our gratitude to the Biology Centre core facilities, namely to the Laboratory of Electron Microscopy, to the Laboratory of Microscopy and Histology, and to the Laboratory of Analytical Biochemistry and Metabolomics. This work was supported by the Horizon Europe ERC MitoSignal project no. 101044951, OP JAK CZ.02.01.01/00/22_008/0004575 RNA for therapy, Co-Funded by the European Union and by Czech Science Foundation project no.23-07370S to AZ. We acknowledge the BC CAS core facility LEM supported by the Czech-BioImaging large RI project (LM2023050 and OP VVV CZ.02.1.01/0.0/0.0/18_046/0016045 funded by MEYS CR) for their support with obtaining scientific data presented in this paper.
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M.K. and E.D. performed the experiments and analyzed the data. M.M. performed the mass spectrometry analyses. B.P. contributed to methodology design. A.Z. conceived and supervised the study and acquired funding. A.Z. wrote the first draft of the manuscript, and M.K. and E.D. contributed to subsequent versions and revisions. All authors reviewed and approved the final manuscript.
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Kunzová, M., Doleželová, E., Moos, M. et al. Reversal of ATP synthase is a key attribute accompanying cellular differentiation of Trypanosoma brucei insect forms. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09933-z
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DOI: https://doi.org/10.1038/s42003-026-09933-z
