Reversal of ATP synthase is a key attribute accompanying cellular differentiation of Trypanosoma brucei insect forms

The mitochondrial F_oF₁-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.