Date of Award
12-2009
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Legacy Department
Genetics
Committee Chair/Advisor
Morris, James C
Committee Member
Marcotte , William
Committee Member
Paul , Kimberly
Committee Member
Smith , Kerry
Abstract
Trypanosoma brucei, the causative agent of the disease African sleeping sickness
in humans and nagana in animals, is a scourge of sub-Saharan Africa. There is a
desperate need for more efficacious therapies for the disease; here we describe research
validating T. brucei hexokinase 1 (TbHK1) as a drug therapeutic target for T. brucei
infection and the identification and characterization of novel inhibitors of the enzyme by
both low throughput and high throughput means. Additionally this thesis introduces
efforts at characterizing a second T. brucei hexokinase, focusing mostly on cell biology
and the determination of localization.
The small molecule quercetin (QCN) was tested and found to inhibit recombinant
TbHK1 and spectroscopic study revealed that quercetin binds to TbHK1 proximal to the
active site. Genetic experiments revealed that QCN may be cytotoxic to parasites as a
result of TbHK inhibition. QCN localized to punctate bodies and the flagellum of T.
brucei (consistent with the localization of TbHKs) and over expression of TbHK1 was
partially protective.
In order to identify additional TbHK1 inhibitors, high through-put screening
(HTS) techniques were implemented. HTS of >220,000 compounds yielded 239 primary
inhibitors of TbHK1. Of the 239 hits, 15 were confirmed in validation assays as TbHK1
inhibitors; these compounds had a spectrum of activity against trypanosomes, suggesting
possible therapeutic leads.
In order to initiate characterization of the unknown function of TbHK2,
localization studies of TbHK2 were performed. Immunofluorescence assays were
employed and TbHK2 was found to localize to the parasite's flagellum, a novel finding
that suggests that glycolysis in the parasite may not be limited to glycosomes, organelles
related to peroxisomes that heretofore were considered the cellular compartments of
glycolysis. Immunofluorescence signal was not exclusive to the flagellum but was
observed in the cytosol and glycosomes. To understand the mode of localization, the
TbHK sequences were aligned with known and predicted flagellum proteins and a
conserved flagellum localization peptide signal was identified in the C-termini of TbHK1
and TbHK2. The peptide signal (RAVLAK) was further explored as a flagellum targeting
sequence using fusions to a reporter. These experiments, along with expression and
localization of HA-tagged TbHK2 revealed that TbHK2 associates with the flagellum and
basal body. This unusual localization suggested that TbHK2 may act as an environmental
glucose sensor or perhaps in energy production for the active flagellum.
The appendix is comprised of three chapters. The first appendix section is a
published paper that focuses on AMP dependent protein kinase from T. brucei. I
contributed microscopic expertise (and intellectual input) and am an author on this work.
The second appendix section is a summary of research performed to characterize a library
of lonidamine analogs against TbHK1 and trypanosomes. And lastly, the third appendix
section is the developed assay for testing the viability of T. brucei cells and adaptation for
high throughput screening.
Recommended Citation
Lyda, Todd, "EXPLORING T. BRUCEI HEXOKINASE BIOLOGY: LOCALIZATION AND INHIBITION STUDIES" (2009). All Dissertations. 449.
https://open.clemson.edu/all_dissertations/449