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

The mitochondrial F _o F ₁ -ATP synthase is a reversible nanomachine responsible for generating the majority of cellular ATP by oxidative phosphorylation. In various pathological contexts associated with mitochondrial dysfunction, this enzyme can reverse its function to maintain essential mitochondrial membrane potential at the expense of ATP. This reversal is regulated by the conserved protein Inhibitory Factor 1 (IF1). Here, we demonstrate that ATP synthase reversal also occurs under physiological conditions during the cellular differentiation of the unicellular parasite Trypanosoma brucei , which transitions between insect and mammalian hosts. Differentiation of the insect forms is marked by upregulation of alternative oxidase (AOX) along with decreased expression of trypanosomal IF1 (TbIF1), collectively creating a metabolic environment conducive to ATP synthase reversal. We show that this reversal is key for proper parasite differentiation: parasites lacking TbIF1 efficiently transitioned into the mammalian-infective form. In these TbIF1 knockout parasites, ATP synthase reversal was associated with an increased ADP/ATP ratio, activation of AMP-activated protein kinase, enhanced cellular respiration driven by elevated proline metabolism, and increased mitochondrial and cellular reactive oxygen species (ROS), known signaling molecules. Conversely, parasites with inducible TbIF1 overexpression failed to reverse ATP synthase activity, showed no AMPK activation or ROS elevation, and remained locked in the initial insect stage. Our findings highlight the critical role of TbIF1 downregulation in enabling life cycle progression and underscore the unique function of the ATP synthase/IF1 axis in cellular signaling.