Date of Award
Doctor of Philosophy (PhD)
Genetics and Biochemistry
Meredith T. Morris
The kinetoplastid parasite Trypanosoma brucei is responsible for both human African trypanosomiasis (HAT) and the wasting disease nagana found in cattle. Unique to kinetoplastids are the specialized peroxisomes, named glycosomes, which compartmentalize the first several steps of glycolysis and gluconeogenesis, nucleotide sugar biosynthesis, and many other metabolic processes. There are many studies surrounding the heterogeneity and complexity of glycosomes as well as how these organelles proliferate and import their proteins. Here, I first explored new methods to analyze glycosome heterogeneity by flow cytometry. The advancement of flow cytometry has yielded methods that enable the identification of vesicles between 30-1000 nm in diameter. I adapted these techniques for the identification of glycosome populations by flow cytometry and the isolation of distinct populations via organelle sorting. With this technology, I detected populations of glycosomes (200 nm) harboring different amounts of a fluorescent glycosome protein reporter. I used a cell sorter to sort glycosomes based on their eYFP expression. T. brucei is unique in that they have two Pex13s, Pex13.1 and Pex13.2, whereas other eukaryotes have a single Pex13, and together, Pex13.1, Pex13.2, and Pex14 comprise the glycosome import complex. Phosphoproteomics reveal that Pex13.2 is phosphorylated and here I analyzed the role of this phosphorylation on both import complex formation and glycosomal protein import. Using Pex13.2 phosphorylation mutants revealed that the phosphorylation state of Pex13.2 does not impact import complex binding or the translocation of matrix proteins. However, Pex13.2 phosphorylation may affect import complex size. Additionally, I examined the activity and protein expression of gluconeogenic enzyme fructose 1,6-bisphosphatase (FBPase) between cell strains. FBPase is localized to the glycosome and recent metabolic labeling experiments revealed the gluconeogenesis is active in both stages of T. brucei. Here, I showed that FBPase is regulated in an unexpected manner. Under low glucose conditions the enzyme activity is undetectable, but in high glucose conditions activity levels are high. Also, I revealed that this activity pattern is both density dependent and strain dependent. Because glycosomes are essential and parasite specific, understanding their biology is critical for development of therapeutics.
Wilkinson, Christina L., "Glycosome Heterogeneity, Import Complex, and Metabolism in Trypanosoma brucei" (2021). All Dissertations. 2817.