Trypanosoma brucei Serine Palmitoyltransferase: Roles In De Novo Sphingolipid Biosynthesis, Life Cycle, and Lipid Raft FormationPublic Deposited
The kinetoplastid protozoan <em>Trypanosoma brucei</em> is the causative agent of African trypanosomiasis, affecting humans, and Nagana disease, affecting cattle, prevalent in regions of sub-Saharan Africa. Our lab demonstrated that the flagellum of <em>T. brucei</em> is enriched in lipid rafts, membrane areas composed of sphingolipids, sterols, and proteins that serve as signal transduction platforms for a variety of cellular processes. In most organisms, sphingolipids and their metabolites are critical for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts. However, recent studies in trypanosomes point to the dispensability of sphingolipids in some life cycle stages of Leishmania, a related kinetoplastid. The enzyme serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid synthesis. To determine the requirements for sphingolipid synthesis in the insect life cycle stage of <em>T. brucei</em>, I targeted the function of <em>T. brucei</em> SPT by RNA interference or treatment with a potent SPT2 inhibitor myriocin. I found that TbSPT2 is required for viability of <em>T. brucei</em>. Mass spectrometry revealed that the parasite contained inositol phosphorylceramides, a sphingolipid common to other kinetoplastids but not previously observed in these parasites, and that their levels were significantly reduced upon TbSPT2 RNAi inhibition or myriocin treatment. Sphingolipid reduction resulted most notably in aberrant cytokinesis, characterized by incomplete cleavage furrow formation, delayed kinetoplast segregation, and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids may act as second messengers regulating cellular proliferation and completion of cytokinesis. However, sphingolipid biosynthesis did not appear to be essential flagellar membrane trafficking and the association of the flagellar membrane protein calflagin with DRMs. Overall, my studies clarified the differences and similarities in cellular requirements for sphingolipid synthesis between kinetoplastids as well as other eukaryotes. I also initiated studies to determine the protein composition of lipid rafts and the lipid composition of the <em>T. brucei</em> flagellum to further understand lipid rafts in this parasite.
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