Date of Award
Doctor of Philosophy (PhD)
Biochemistry and Molecular Biology
The protozoan pathogen, Trypanosoma brucei, is the causative agent of sleeping sickness in humans and nagana in livestock in sub-Saharan Africa. T. brucei cycles between tsetse fly and mammalian hosts, and it is adapted to survive in diverse host tissues. Variant Surface Glycoprotein (VSG) plays a key role in immune evasion in the mammalian host. The VSG membrane anchor requires two myristates, 14-carbon saturated fatty acids (FAs) that are scarce in the host. T. brucei can synthesize FAs de novo, but also readily takes up exogenous FAs, despite lacking homologs to fatty acid uptake proteins found in other organisms.
Previous work in our lab found that Triacsin C, an inhibitor of acyl-CoA synthetases (ACSs), inhibited the uptake of fluorescent FAs and was toxic to T. brucei. I hypothesized that Grassofermata (GF), an inhibitor of FA transport proteins (FATPs), might also affect T. brucei, as FATPs have intrinsic ACS activity. I found that both mammalian bloodstream forms (BFs) and tsetse fly midgut procyclic forms (PFs) of T. brucei internalized fluorescently-labeled FAs (BODIPIY-FAs). Treatment with GF decreased BODIPY-FA uptake as measured by fluorescence microscopy and flow cytometry, though GF was unable to completely inhibit BODIPY-FA uptake. The residual BODIPY-FA uptake observed under GF inhibition was not due to endocytic mechanisms.
Treatment with GF also killed both BF and PF T. brucei, and cell death was dependent on GF concentration as well as treatment time. Addition of unlabeled C12:0 and C16:0 FAs to GF-treated cells was unable to “rescue” cell growth. Finally, the GF concentrations required to inhibit BODIPY-FA uptake (EC50s of ~70–100 µM) were much higher than that for Triacsin C inhibition (2–8 µM). Importantly, the EC50s for BODIPY-FA uptake inhibition by GF were higher than that for cell killing by GF (EC5048hr of 4-17 µM). Based on these results, we conclude that GF killing of T. brucei does not seem to be a result of inhibiting FA uptake and/or ACSs. Instead, GF may be affecting other known targets in the cell, such as ADP-ribosylation factor (ARF) and its accessory factors. Future studies will be needed to confirm the mode of action of GF in T. brucei.
Parman, Kristina Marie, "Characterization of the Effects of The Pyrazolopyrimidine Inhibitor Grassofermata (NAV-2729) in the Eukaryotic Pathogen Trypanosoma Brucei" (2023). All Dissertations. 3527.
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