The African trypanosome Trypanosoma brucei is a single-celled eukaryotic protozoan parasite with a single cilium/flagellum. It cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. For the first part of the work we describe here, we focus on T. brucei cilium and its lipid rafts. Lipid rafts are liquid order microdomains enriched in sterols, sphingolipids and acylated proteins. Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. We tested whether ciliary sterol enrichment results from selective ciliary partitioning of specific sterol species or to general enrichment of all sterols. While all sterols are enriched in the cilium, cholesterol is especially enriched. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl--cyclodextrin disrupts both the membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on ciliary sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment. As a follow-up on the sterol work, we determined the lipid raft proteomes of the two life cycle stages of T. brucei. Although both the proteomes were enriched in flagellar and palmitoylated proteins, majority of the proteins in the lipid raft proteomes were either stage specific or were only found in the lipid raft proteome of one life cycle stage. Consistent to our results from the life cycle stage specific lipid raft biology, we found a life cycle stage specific lipid raft protein profile as well. Our findings establish T. brucei cilium as the lipid raft enriched hub for sterols, sphingolipids and palmitoylated proteins involved in signaling and virulence. In the final work that focuses on protein depalmitoylation, we characterized the single acyl protein thioesterase (APT) in T. brucei, which is most similar to the human APT2. It was identified in the procyclic lipid raft proteome of T. brucei. Indeed, the biochemical properties of the TbAPT and human APT 2 are similar. Both knockdown and overexpression of the TbAPT resulted in decreased parasite virulence in mice, despite there being no notable in vitro phenotype. Since TbAPT is a single acyl-thioesterase in an organism with 12 palmitoyl acyltransferases (PATs), it is likely that a defect in proper protein palmitoylation homeostasis mediates the virulence defect. We determined a list of putative TbAPT substrates using acyl biotin exchange assay followed by mass spectrometry. The most surprising revelation involved the high confidence possibility of variant surface glycoprotein (VSG) to be a putative TbAPT substrate. Through several ongoing studies to unravel the role of TbAPT in mediating virulence, we hope to bridge the gap in the understanding of dynamic palmitoylation in kinetoplastids.

Last modified

• 03/28/2018

Creator

• Aabha Sharma

DOI

• https://doi.org/10.21985/N2JX0S

Subject

• Driskill Graduate Training Program in Life Sciences

Keyword

• Lipid rafts
• Sterols
• Trypanosome
• Palmitoylation

Date created

• 2017-01-01

Resource type

• Dissertation

Rights statement

• In Copyright