Mitochondrial ATP synthase is a reversible nanomotor synthesizing or hydrolyzing ATP depending on the potential across the membrane in which it is embedded. In the unicellular parasite Trypanosoma brucei, the direction of the complex depends on the life cycle stage of this digenetic parasite: in the midgut of the tsetse fly vector (procyclic form), the FoF1-ATP synthase generates ATP by oxidative phosphorylation, whereas in the mammalian bloodstream form, this complex hydrolyzes ATP and maintains mitochondrial membrane potential (ΔΨm). The trypanosome FoF1-ATP synthase contains numerous lineage-specific subunits whose roles remain unknown. Here, we seek to elucidate the function of the lineage-specific protein Tb1, the largest membrane-bound subunit. In procyclic form cells, Tb1 silencing resulted in a decrease of FoF1-ATP synthase monomers and dimers, rerouting of mitochondrial electron transfer to the alternative oxidase, reduced growth rate and cellular ATP levels, and elevated ΔΨm and total cellular reactive oxygen species levels. In bloodstream form parasites, RNAi silencing of Tb1 by ∼90% resulted in decreased FoF1-ATPase monomers and dimers, but it had no apparent effect on growth. The same findings were obtained by silencing of the oligomycin sensitivity-conferring protein, a conserved subunit in T. brucei FoF1-ATP synthase. However, as expected, nearly complete Tb1 or oligomycin sensitivity-conferring protein suppression was lethal because of the inability to sustain ΔΨm. The diminishment of FoF1-ATPase complexes was further accompanied by a decreased ADP/ATP ratio and reduced oxygen consumption via the alternative oxidase. Our data illuminate the often diametrically opposed bioenergetic consequences of FoF1-ATP synthase loss in insect versus mammalian forms of the parasite.

• Keywords
• ATP synthase, ATPase, Trypanosoma brucei, alternative oxidase, bioenergetics, electron transport, mitochondria, mitochondrial membrane potential, oxidative phosphorylation, respiration,
• MeSH
• Adenosine Triphosphate genetics   metabolism   MeSH
• Cell Cycle * MeSH
• Energy Metabolism * MeSH
• Membrane Potential, Mitochondrial MeSH
• Mitochondria genetics   metabolism   MeSH
• Proton-Translocating ATPases deficiency   metabolism   MeSH
• Protozoan Proteins genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Proton-Translocating ATPases MeSH
• Protozoan Proteins MeSH

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.

• MeSH
• Adenosine Triphosphate biosynthesis   MeSH
• Cell Differentiation drug effects   MeSH
• Cell Respiration drug effects   MeSH
• Cell Line MeSH
• Electrons MeSH
• Glucose pharmacology   MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• Metabolic Networks and Pathways drug effects   MeSH
• Metabolomics * MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondria drug effects   metabolism   MeSH
• Oxidation-Reduction MeSH
• Oxidoreductases metabolism   MeSH
• Proline metabolism   MeSH
• Proteome metabolism   MeSH
• Protozoan Proteins metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Signal Transduction MeSH
• Transcriptome genetics   MeSH
• Electron Transport drug effects   MeSH
• Trypanosoma brucei brucei drug effects   genetics   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• alternative oxidase MeSH Browser
• Glucose MeSH
• Mitochondrial Proteins MeSH
• Oxidoreductases MeSH
• Proline MeSH
• Proteome MeSH
• Protozoan Proteins MeSH
• Reactive Oxygen Species MeSH
• Plant Proteins MeSH

Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

• Keywords
• FoF1 ATPase, Mitochondrion, Phosphonium salt, SDH complex, Succinate dehydrogenase, Trypanosoma brucei,
• MeSH
• Adenosine Triphosphate metabolism   MeSH
• Azides pharmacology   MeSH
• Cell Line MeSH
• Membrane Potential, Mitochondrial drug effects   MeSH
• DNA, Mitochondrial metabolism   MeSH
• Mitochondria drug effects   genetics   metabolism   MeSH
• Organophosphorus Compounds chemistry   pharmacology   MeSH
• Proton-Translocating ATPases metabolism   MeSH
• RNA Interference MeSH
• Succinate Dehydrogenase metabolism   MeSH
• Trypanocidal Agents pharmacology   MeSH
• Trypanosoma brucei brucei cytology   drug effects   growth & development   MeSH
• Trypanosomiasis, African parasitology   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphate MeSH
• Azides MeSH
• DNA, Mitochondrial MeSH
• Organophosphorus Compounds MeSH
• Proton-Translocating ATPases MeSH
• Succinate Dehydrogenase MeSH
• Trypanocidal Agents MeSH
• Calcium MeSH

The highly conserved ADP/ATP carrier (AAC) is a key energetic link between the mitochondrial (mt) and cytosolic compartments of all aerobic eukaryotic cells, as it exchanges the ATP generated inside the organelle for the cytosolic ADP. Trypanosoma brucei, a parasitic protist of medical and veterinary importance, possesses a single functional AAC protein (TbAAC) that is related to the human and yeast ADP/ATP carriers. However, unlike previous studies performed with these model organisms, this study showed that TbAAC is most likely not a stable component of either the respiratory supercomplex III+IV or the ATP synthasome but rather functions as a physically separate entity in this highly diverged eukaryote. Therefore, TbAAC RNA interference (RNAi) ablation in the insect stage of T. brucei does not impair the activity or arrangement of the respiratory chain complexes. Nevertheless, RNAi silencing of TbAAC caused a severe growth defect that coincides with a significant reduction of mt ATP synthesis by both substrate and oxidative phosphorylation. Furthermore, TbAAC downregulation resulted in a decreased level of cytosolic ATP, a higher mt membrane potential, an elevated amount of reactive oxygen species, and a reduced consumption of oxygen in the mitochondria. Interestingly, while TbAAC has previously been demonstrated to serve as the sole ADP/ATP carrier for ADP influx into the mitochondria, our data suggest that a second carrier for ATP influx may be present and active in the T. brucei mitochondrion. Overall, this study provides more insight into the delicate balance of the functional relationship between TbAAC and the oxidative phosphorylation (OXPHOS) pathway in an early diverged eukaryote.

• MeSH
• Mitochondrial ADP, ATP Translocases chemistry   genetics   metabolism   MeSH
• Evolution, Molecular * MeSH
• Oxidative Phosphorylation * MeSH
• Protozoan Proteins chemistry   genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Mitochondrial ADP, ATP Translocases MeSH
• Protozoan Proteins MeSH