Abstract
The mitochondrial (mt) FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF), but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF), which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm). Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1) binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1), but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.
Highlights
Trypanosoma brucei rhodesiense and T. b. gambiense are the flagellated protists responsible for Human African Trypanosomiasis in 36 sub-Saharan African countries where the tsetse fly insect vector resides
Due to the ability of inhibitory factor 1 (IF1) to inhibit the hydrolytic activity of FoF1-ATPase and its potential to selectively impact the viability of the infectious stage of T. brucei, we identified the IF1 homolog in this human pathogen and characterized its effect on the cellular growth and mt physiology in both parasitic life stages
IF1 is widespread throughout the eukaryotic kingdom, with homologs found in plants, yeast, worms and vertebrates
Summary
Trypanosoma brucei rhodesiense and T. b. gambiense are the flagellated protists responsible for Human African Trypanosomiasis in 36 sub-Saharan African countries where the tsetse fly insect vector resides. A striking feature of T. brucei is the ability to rapidly adapt its metabolism while alternating between the glucose-rich blood of a mammalian host and the abundant proline found in the hemolymph and tissue fluids of the blood-feeding tsetse fly [2,3] These different carbon sources dictate a shift in bioenergetics that manifests in the unique architecture and activities of the singular mitochondrion. The enzymatic complexes III and IV pump protons into the mitochondrial (mt) intermembrane space, generating a membrane potential (Δψm) that is coupled to ATP production by the FoF1-ATP synthase [5,6] This rotary molecular machine converts the potential energy of the proton gradient into the chemical energy of ATP when protons are allowed to flow down their energy gradient by passing through the proton pore located in the membrane-embedded Fo domain. This action drives the rotation of the asymmetrical central stalk within the matrix protruding F1 catalytic domain, which undergoes three sequential conformational states that result in the synthesis of ATP
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