Date of Award
12-2016
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Legacy Department
Chemistry
Committee Member
Dr. Kenneth Christensen, Committee Chair
Committee Member
Dr. Meredith Morris
Committee Member
Dr. Jeffery Anker
Committee Member
Dr. George Chumanov
Abstract
Trypanosoma brucei is a unicellular kinetoplastid protozoan that is the causative agent of Human African Trypanosomiasis, or Sleeping Sickness. Combating these protozoan pathogens presents an ongoing challenge due to their efficient immune evasion strategies, lack of effective vaccines, poor drugs and emerging drug resistance, and the general lack of knowledge of their unique biological processes. Among one of the most important biological pathways for kinetoplastid protozoans is the synthesis of ATP from glucose within glycosome organelles. Glycosomes are related to mammalian peroxisomes, but differ in having a fully compartmentalized glycolysis pathway in addition to basic peroxisomal functions. While equivalent processes and organelles have been thoroughly studied through live cell imaging in other organisms, similar studies of T. brucei have only recently made significant progress and many fluorescent-based techniques have not yet been applied in kinetoplastid studies. In order to increase our understanding of trypanosome biology, we have developed quantitative fluorescence-based sensors for sub cellular analysis of metabolic pathways in T. brucei. The pH of the glycosomal compartment was quantified with a probe consisting of a peptide encoding a peroxisomal targeting sequence attached to fluorescein. When incubated with living cells, the probe is internalized within glycosomes and allowed for the quantification of pH through ratiometric analysis of fluorescein emission at 495 nm and 430 nm excitation. Using this probe, we were able to measure the physiological pH (~7.4) of glycosomes under standard culture conditions, as well as discover a pH acidification response (~ 6.8) to nutrient starvation. This starvation response is dependent on Na+ and ATP and regulated independently form the cytosol. The use of transporter and exhcnager inhibitors suggest that V-ATPase and Na+/H+ exchangers are responsible for glycosome pH regulation. We also adapted a ratiometric forster resonance energy transfer (FRET) protein construct (FLIPGlu) consisting of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) FRET pairs flanking a periplasmic binding protein. This protein changes conformation upon binding to glucose, resulting in a measureable change in FRET ratio. FLIPGlu was expressed in T. brucei glycosomes, allowing for the dynamic quantification of glucose. Trypanosomes were found to maintain 230 ± 50 and 530 ± 50 µM glucose in the insect and mammalian life stage respectively. Glucose starvation resulted in a decrease in glycosome glucose levels (~40 ± 10 µM) over 30 minutes but recovers upon reintroduction of glucose. The work presented here fulfills an important need for the implementation of powerful fluorescent techniques to study Trypanosome metabolic pathways. This can greatly advance the understanding of kinetoplastid biology and lead to insights in evolutional divergence of trypanosome biology and therapeutic treatments. While the probes studied here were developed to specifically study pH, and glucose levels relating to glycolysis and glycosomes, these platforms can be modified to cover a wide range other biological parameters, analytes, and organelles.
Recommended Citation
Lin, Sheng, "FLUORESCENT METHODS FOR INVESTIGATING METABOLIC PROCESSES IN TRYPANOSOMA BRUCEI" (2016). All Dissertations. 1833.
https://tigerprints.clemson.edu/all_dissertations/1833