Discussion

The Trypanosoma brucei genome contains two glucose transporter gene types, each present in five copies organized in an array. Trypanosoma hexose transporter 1 (THT1) dominates in the bloodstream stage, whereas THT2 is procyclic specific. These transporters are thought to be the sole route of glucose supply for glycolysis in bloodstream form cells, while the need for a distinct transporter in the insect-stage has remained mysterious. We prepared RNAi constructs targeting each group of transporter genes specifically, or both simultaneously. We also tagged and localised a copy of each group to facilitate monitoring of knockdown; both proteins were localised to the plasma membrane, as expected. In the bloodstream form, THT1 knockdown lead to a growth defect, but the defect was more severe following THT1-THT2 double knockdown. In contrast, knockdown of both transporters in the procyclic stage resulted in only a minor growth defect. These outcomes are consistent with an exquisite dependence on glycolysis for ATP production in bloodstream-form cells; glucose is considered to be the only carbon source used by this life cycle stage. Somewhat surprisingly, the severe growth defect in bloodstream-form cells was rescued by supplementation of glycerol in the growth media. Subsequent metabolomics analysis demonstrated that these bloodstream-form cells take up and metabolise glycerol, using it for the synthesis of fructose 6-phosphate, and to fuel the tricarboxylic acid cycle. To our knowledge, this is the first observation of gluconeogenesis in bloodstream-form T. brucei. Glycerol may be a physiological substrate for gluconeogenesis in natural host tissues.