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 gluco-neogenesis 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 C-13-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Delta ppdk/Delta pepck), but not in the single Delta ppdk and Delta pepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeo-genesis, since the death of the Delta ppdk/Delta pepck 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 (Delta fbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of C-13-enriched proline into G6P. In addition, the Delta fbpase 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 Delta fbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Delta fbpase 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 Delta fbpase mutant. Keyword