Date of Award

5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Committee Member

Dr. George Chumanov, Committee Chair

Committee Member

Dr. Kenneth Christensen

Committee Member

Dr. James Morris

Committee Member

Dr. Daniel Whitehead

Abstract

Members of the class Kinetoplastea including Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. cause diseases endemic in rural regions of South America, Sub‐Saharan African and the Eastern Asian continent, effecting hundreds of millions of people and livestock. Existing treatments are associated with high toxicity and rates of resistance, are expensive to produce, and are difficult to administer in rural areas. To develop additional treatment strategies, we must better illuminate the pathways amenable for anti kinetoplastid treatments. One pathway susceptible to drug intervention is glucose metabolism, which in kinetoplasts takes place in glycosomes that are specialized organelles related to mammalian peroxisomes. Disruption of glycosome function is hypothesized to lead to cell death in the pathological bloodstream form of T. brucei as they obtain all cellular ATP via glycosome metabolism. To explore glucose import, and consumption mechanisms in T. brucei we deployed a series of recombinant fluorescent protein biosensors that specifically detect glucose moieties. Biosensors were expressed in T. brucei, and targeted to the cytosol or glycosomes allowing for real time monitoring of intracellular and intraglycosomal glucose concentrations. Using flow cytometry to monitor changes in sensor fluorescence, bloodstream form T. brucei cytosolic and glycosomal glucose were measured as 1.9 ±0.6 mM and 3.5 ± 0.5 mM respectively in response to glucose levels similar to blood (~5mM). Higher glycosomal glucose versus the surrounding cytosol suggests active transport of glucose across the glycosomal membrane, a process that was assumed to occur via passive transport. Monitoring biosensor response in trypanosomes accurately via microscopy is very difficult due to high motility and flagellar undulation. To monitor dynamics in intracellular biosensor response we adapted a microfluidic device which mechanically traps parasites, allowing for continuous imaging of cells under constant perfusion conditions. We found that single trypanosome glucose responses were consistent with bulk glucose measurements, cells also responded in a dose dependent manner when perfused against different glucose concentrations. Microfluidic trapping of T. brucei allows continuous imaging of single cellular dynamics which were previously not possible to image. To identify small molecules that inhibit glucose uptake into parasites we adapted a high throughput screening assay utilizing the fluorescent glucose sensor as a score of glucose uptake inhibition. A pilot screen of 400 compounds identified two novel compounds that inhibit glucose uptake in trypanosome parasites with EC50s of 700nM and 5000nM respectively, one compound exhibited good killing (IC50 5uM) against infectious form parasites. To build upon the success of the pilot screen, 25,000 compounds were analyzed, from this library 57 compounds were identified, 40 of which kill infectious form trypanosomes with an IC50 value lower than 5uM. As predicted, trypanosome specific glucose uptake inhibitors identified in our screen exhibit potent anti‐trypanosome killing activity. The findings herein describe methods that help fill in the gaps in kinetoplastid glycosome glucose transport and metabolism. Using these methods, we have characterized glycosomal glucose transport, monitored single trypanosomes via fluorescent microscopy, and identified glucose uptake inhibitors specific to T. brucei. Although the methods here are limited to glucose measurements, they are amenable to studying a wide range of biologically relevant analytes with the broad pallet available fluorescent biosensors, in organelles of the parasitic kinetoplasts.

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