Abstract
The sleeping sickness parasite Trypanosoma brucei has a complex life cycle, alternating between a mammalian host and the tsetse fly vector. A tightly controlled developmental programme ensures parasite transmission between hosts as well as survival within them and involves strict regulation of mitochondrial activities. In the glucose-rich bloodstream, the replicative ‘slender’ stage is thought to produce ATP exclusively via glycolysis and uses the mitochondrial F1FO-ATP synthase as an ATP hydrolysis-driven proton pump to generate the mitochondrial membrane potential (ΔΨm). The ‘procyclic’ stage in the glucose-poor tsetse midgut depends on mitochondrial catabolism of amino acids for energy production, which involves oxidative phosphorylation with ATP production via the F1FO-ATP synthase. Both modes of the F1FO enzyme critically depend on FO subunit a, which is encoded in the parasite’s mitochondrial DNA (kinetoplast or kDNA). Comparatively little is known about mitochondrial function and the role of kDNA in non-replicative ‘stumpy’ bloodstream forms, a developmental stage essential for disease transmission. Here we show that the L262P mutation in the nuclear-encoded F1 subunit γ that permits survival of ‘slender’ bloodstream forms lacking kDNA (‘akinetoplastic’ forms), via FO-independent generation of ΔΨm, also permits their differentiation into stumpy forms. However, these akinetoplastic stumpy cells lack a ΔΨm and have a reduced lifespan in vitro and in mice, which significantly alters the within-host dynamics of the parasite. We further show that generation of ΔΨm in stumpy parasites and their ability to use α-ketoglutarate to sustain viability depend on F1-ATPase activity. Surprisingly, however, loss of ΔΨm does not reduce stumpy life span. We conclude that the L262P γ subunit mutation does not enable FO-independent generation of ΔΨm in stumpy cells, most likely as a consequence of mitochondrial ATP production in these cells. In addition, kDNA-encoded genes other than FO subunit a are important for stumpy form viability.
Highlights
The parasitic protist Trypanosoma brucei undergoes a complex life cycle involving stages within the mammalian bloodstream and its tsetse fly vector
Little is known about the metabolic requirements of stumpy forms, but studies have demonstrated an increase in the abundance of many mitochondrial proteins in the stumpy life cycle form compared to the slender bloodstream form (BSF), including subunits of the mitochondrial respiratory complexes and key mitochondrial metabolic enzyme activities such as pyruvate dehydrogenase, α-ketoglutarate (α-KG) dehydrogenase, acetate:succinate CoA-transferase (ASCT) and succinyl-CoA synthetase (SCoAS) [9,10,11,12]
In order to be able to investigate the requirement for functional kDNA in the differentiation of T. brucei from slender to stumpy form, we replaced one allele of the nuclear-encoded F1FOATPase subunit γ with a version with the L262P mutation (L262Pγ) in the pleomorphic cell
Summary
The parasitic protist Trypanosoma brucei undergoes a complex life cycle involving stages within the mammalian bloodstream and its tsetse fly vector. The emergence of cell cycle-arrested stumpy forms prevents parasitaemia increasing further, prolonging host survival, and results in the characteristic waves of parasitaemia seen in bloodstream infections in rodents. This density dependent differentiation has been shown to be induced by a stumpy induction factor (SIF) via a form of quorum sensing [2]. PCF are able to generate ATP using mitochondrial energy production pathways, involving both oxidative and substrate-level phosphorylation [3,4,5,6]. ATP production in the slender BSF is thought to solely involve non-mitochondrial glycolysis, utilising the glucose-rich environment found within the mammalian bloodstream [7,8]. Cytochromes have not been detected in stumpy forms [10], but the presence of an abbreviated oxidative phosphorylation pathway consisting of respiratory complexes I (cI; NADH:ubiquinone oxidoreductase) and V (F1FO-ATP synthase) and the trypanosome alternative oxidase (AOX) has been proposed [14]
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