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FRET Cytometric Method for High Throughput Screening of Potential Metabolic Inhibitors in Trypanosoma brucei
Author(s) -
Voyton Charles Michael,
Morris Meredith T,
Ackroyd P. Christine,
Morris James C,
Christensen Kenneth A
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb143
Subject(s) - trypanosoma brucei , biology , biochemistry , hexose , cytosol , glucose transporter , glycolysis , flow cytometry , high throughput screening , microbiology and biotechnology , metabolism , enzyme , gene , insulin , endocrinology
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 that under normal growth conditions glycosomal glucose is nearly ten times that of the cytosol. Cellular glucose concentrations were monitored in the presence of candidate glycolytic and hexose transporter inhibitors allowing for inhibition efficacy to be scored. Using this methodology we have now developed high throughput screening (HTS) assays for inhibitors of glucose metabolism and hexose transport in PCF and BSF parasites. The screen allows scoring of 250 test compounds per hour and a robust Z’‐values of 0.56 and 0.74 for BSF and PCF assays respectively. We have demonstrated that flow cytometric analysis of FRET biosensors can serve as a potent platform for analysis of metabolite flux in live T. brucei as well as implementing this strategy in a high throughput screen for molecules that inhibit glucose metabolism and transport in live T. brucei . Support or Funding Information R21AI105656 from NIH and Brigham Young University