Abstract by Jongsu Choi
Chemistry and Biochemistry
Improvement of the glucose FRET sensor in Trypanosoma brucei using brighter fluorescence protein variants and codon optimization
Trypanosoma brucei, the causative agent of human African trypanosomiasis (sleeping sickness), is a kinetoplastid parasite transmitted via the tsetse fly in sub-Saharan Africa. In the human host, the sole source of ATP production in T. brucei is the peroxisome like-organelle called the glycosome that compartmentalizes essential metabolic processes such as glycolysis. Glycolysis is an indispensable component for survival of T. brucei in the host and the inhibition of glycolysis is a viable therapeutic target of trypanosomiasis. Using a glucose-sensitive Förster resonance energy transfer (FRET) sensor, we have measured both cytosolic and glycosomal glucose concentrations inside of T. brucei. However, both the overall fluorescence intensity and glucose dynamic range for the FLIPglu-600 sensor limit its usefulness. Here we report substantial improvements in the glucose sensor in vitro and in trypanosomes using brighter fluorescent protein variants and codon optimization for expression. We will also demonstrate how the improved performance is utilized in both flow cytometry and fluorescence microscopy for glucose measurements in live T. brucei.