Gluconeogenesis is essential for trypanosome development in the tsetse fly vector

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.
Author summary Trypanosoma brucei, the parasite responsible for sleeping sickness in humans, is transmitted by the tsetse fly that primarily uses amino acids for its energy production. In the glucose-free environment encountered between the insect blood meals, T. brucei needs to produce through gluconeogenesis glucose 6-phosphate, a key precursor for several essential metabolic pathways. We have shown here that two key gluconeogenic steps, which produce phosphoenolpyruvate and fructose 6-phosphate, respectively, are performed by redundant enzymes (PPDK and PEPCK for phosphoenolpyruvate production; FBPase and a yet unknown enzyme for fructose 6-phosphate production), which highlights the importance of this metabolic pathway for the insect stages of the parasite. Interestingly, deletion of the parasite FBPase gene abolished both the colonisation of the insect salivary glands and the in vitro differentiation of the epimastigote forms into the mammalian infective form of the parasite. Altogether, these data demonstrate for the first time that gluconeogenesis is essential for development of T. brucei in its insect vector and that early development stages of the parasite present in the tsetse midgut are not affected by the absence of FBPase, probably by developing an alternative yet unknown approach to produce fructose 6-phosphate.