Author Date

2021-06-18

Degree Name

Department

Chemistry and Biochemistry

College

Physical and Mathematical Sciences

Defense Date

2021-06-08

Publication Date

2021-06-18

First Faculty Advisor

Kenneth A. Christensen

First Faculty Reader

Christine Ackroyd

Honors Coordinator

Walter Paxton

Keywords

FRET biosensor, Trypanosoma brucei, flow cytometry, glucose metabolism, multiplex, barcoding

Abstract

Kinetoplastid parasites are a significant public health issue in some tropical and subtropical regions of the world. Kinetoplastid parasites all require glycolysis for survival, with host glucose key for ATP production. One such parasite, Trypanosoma brucei, exclusively metabolizes glucose in its bloodstream form. Trypanosomal glycolysis is unique because it displays unconventional structural features. Hence, glucose metabolism has been studied extensively in T. brucei and is a therapeutic target in kinetoplastid parasites.The lack of in vivo analytical techniques for measuring vital glycolytic metabolites in situ has restricted the ability of researchers to test, with high sensitivity and specificity, the essential roles glycolytic metabolites play in both parasite differentiation and regulation of metabolism. Using endogenously expressed Förster resonance energy transfer (FRET) biosensors, we have been able to track in vivo glucose concentrations via flow cytometry in cultured T. brucei parasites, which has enabled unprecedented analysis of the dynamics of intracellular glucose under changing extracellular conditions, including a screening assay for potential glucose uptake inhibitors. We are currently expanding our FRET biosensor suite to measure glycolytic metabolites and simultaneously screen for specific glycolytic inhibitors throughout the metabolic pathway. We expect to use biosensors for glucose, ATP, pH, and pyruvate. However, these sensors typically use the same fluorescent proteins, making it extraordinarily difficult to monitor multiple biosensors in the flow cytometer. To overcome this limitation, we present a cellular barcoding method in which multiple cell lines expressing a different biosensor have unique labels to separate individual sensor responses via flow cytometry. Each cell line is distinguishable from one another using a cell surface staining scheme or barcode. Using this approach, we demonstrated the separation of up to four unique populations, thus allowing the analysis of multiple analytes in a single screening assay. As proof of principle, we demonstrated the barcoding of two cell lines expressing cytosolic glucose or ATP sensors and simultaneously measured cytosolic glucose and ATP. We found that cellular barcoding does not interfere with parasite cell viability, and cell populations remained distinguishable over time. We also showed that the individual FRET biosensor responses remain unaffected by barcoding. Finally, we demonstrated that this method could be used in a high-throughput screening assay for T. brucei and other kinetoplast parasites to identify compounds that inhibit glycolysis.