Environmental Regulation of Fatty Acid Synthesis in Trypanosoma brucei
We have found that Acetyl-CoA Carboxylase (ACC) the first step in fatty acid synthesis, is regulated by phosphorylation in response to the supply of environmental lipids in the procyclic form of T. brucei, which is found in the midgut of its tsetse fly insect vector. When environmental lipids are high, ACC is phosphorylated, which turns ACC activity off. When environmental lipids are low, ACC is dephosphorylated, which activates ACC. Our hypothesis is that when environmental lipids are high, the parasite can rely on uptake for its fatty acid needs, and so it turns off fatty acid synthesis to conserve energy. When environmental lipids are insufficient, ACC is turned on so the parasite can synthesize the fatty acids it can’t get from the environment.
Unanswered Questions: Who is the ACC kinase? What amino acids on ACC are phosphorylated? How does the parasite sense the levels of environmental lipids?
Defining Mechanism(s) of Fatty Acid Uptake in Trypanosomatids
Trypanosomatids readily take up fatty acids from their environment, yet little is known about the molecular mechanisms involved nor the relative importance of this process in the host-parasite relationship. A search of the genome revealed that trypanosomatids lack the typical fatty acid transport proteins found in other eukaryotes, suggesting a streamlined approach to fatty acid uptake. We have developed a fluorescence-based fatty acid uptake assay and are using it to biochemically characterize fatty acid uptake in three different trypanosomatids, the insect and bloodstream form stages of Trypanosoma brucei, the causative agent of African sleeping sickness; Crithidia fasciculata, a monoxenous pathogen of mosquitoes; and Bodo saltans, a free-living trypanosomatid. In addition, we have compiled a list of genes form the genome that are potentially involved in fatty acid uptake, and are systematically knocking-down their function using RNA interference to assess their role in fatty acid uptake.
Unanswered Questions: What is mediating fatty acid uptake in trypanosomatids? How is fatty acid uptake regulated? How are exogenous fatty acids trafficked and partitioned after bring taken up?
3. Myristate Utilization
T. brucei bloodstream forms have a particular reliance on myristate, a 14-carbon saturated fatty acid. The major surface protein, Variant Surface Glycoprotein (VSG), is anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI)-anchor that contains two myristates. We hypothesize that fatty acid synthesis is required in the bloodstream form to supply sufficient myristate for VSG GPI-anchor myristoylation. A corollary to this hypothesis is that the VSG GPI-anchor myristoylation pathway gets priority status in using newly-synthesized myristate, over other myristate-utilization pathways. We are examining the partitioning of myristate between competing myristate-utilizing pathways and the how this partitioning may be controlled.
Unanswered Questions: How is newly synthesized myristate partitioned between GPI-myristoylation, phospholipid synthesis, and protein myristoylation pathways? Is myristate taken up from the environment trafficked differently than other fatty acids? What fraction of the VSG GPI-anchor myristates are synthesized vs. acquired from host blood?
4. Lipid Storage
Intracellular lipid storage is mediated by lipid droplets, spherical dynamic organelles bound by a membrane monolayer. To date, we have established using fluorescence microscopy and Nile Red staining that both T. brucei and C. fasciculata have lipid droplets, suggesting these are generally common features of Trypanosomatids. Interestingly, a search of the genome shows no obvious homologs to the major known lipid droplet proteins that have been described in other organisms such as yeast and mammals. This suggests that Trypanosomatids may have a more basal, evolutionarily older lipid droplet machinery. The goal of this project is to biochemically purify lipid droplets in Crithidia and T. brucei and then use mass spectrometry to identify the protein components of trypanosomatid lipid droplets.
Unanswered Questions: How does the lipid droplet machinery of Trypanosomatids compare to yeast and mammalian lipid droplets? How are the numbers and size of lipid droplets regulated? What cellular role(s) do lipid droplets serve in Trypanosomatids and how does this vary with the life history of different species?