FRET Cytometric Method for High Throughput Screening of Potential Metabolic Inhibitors in Trypanosoma brucei

The infectious form of Trypanosoma brucei survives in the mammalian host by converting blood glucose into ATP in a specialized peroxisome-like organelle called the glycosome. Compartmentalization of glycolysis and the requirement of glucose as the sole carbon source for parasite survival make glucose metabolism and hexose transport in T. brucei an important area of investigation and a promising pathway for targeted anti-parasite therapy. Here we describe the application of a genetically encoded fluorescent biosensor (FLII12Pglu-600u) which we endogenously expressed in the cytosol of the infectious bloodstream form (BSF) and in the glycosome of the insect intermediate procyclic form (PCF) allowing for quantification of cytosolic and glycosomal glucose concentration. This methods utilizes flow cytometry for high throughput analysis, as opposed lower throughput microscopy methods typically used with fluorescent protein sensors. Using this method we have measured glucose flux in live BSF and PCF cells showing...