The Role of Fatty Acid Metabolism in the Pathogenesis of Trypanosoma brucei

Trypanosoma brucei is the protozoan parasite that causes African Sleeping Sickness in humans and nagana, a wasting disease in cattle. T. brucei completes its life cycle in two hosts, mammals and the tsetse fly insect vector. Due to the geographical restriction of the tsetse fly, the disease is endemic in sub-Saharan Africa. Both the insect and mammalian forms of the parasite need fatty acids to anchor their surface proteins. We worked on three projects on fatty acid metabolism and its role in immune evasion strategies of T. brucei. First, we assessed the role of T. brucei surface proteins in fatty acid uptake. We used biotin-labeling of the cell surface proteins followed by digestion with proteinase K. Then we performed BODIPY-fatty acid uptake assays. We found no evidence for the involvement of surface proteins in fatty acid uptake in the insect and mammalian bloodstream forms (BSFs) of the parasite. Second, we assessed the dynamics of surface Variant Surface Glycoprotein (VSG) under downregulation of fatty acid synthesis. VSG helps T. brucei BSFs evade host immune response, and the VSG glycosylphosphatidylinositol (GPI) membrane anchor and its two myristates are thought to play a key role in VSG mobility and trafficking. The role of fatty acid synthesis (FAS) in VSG function is unknown, but it is likely needed to maintain VSG GPI-anchor myristoylation. We used RNAi-mediated depletion of Acetyl-CoA Carboxylase (ACC), the first enzyme in the FAS pathway. To determine the effect of ACC RNAi on VSG dynamics, we assessed VSG half-life by surface biotinylation and streptavidin blotting. We found that ACC RNAi decreased surface VSG half-life and increased biotinylated VSG internalization. We also found that ACC RNAi affected endocytic trafficking. ACC RNAi had no apparent effect upon receptor-mediated endocytosis of transferrin, but did increase Concanavalin A uptake at five min. The effect of ACC RNAi on fluid-phase endocytosis depended upon the marker, with FM 4-64 endocytosis increased while dextran (500S) endocytosis showed no change. Altogether, our observations indicate that FAS is required for normal VSG dynamics and endocytosis. The third project was to identify the GPI-anchor sn1-remodelase in BSF T. brucei. VSG GPIs are initially synthesized with longer fatty acids, but before incorporation into the VSG precursor, GPI anchors are remodeled with the two longer fatty acids being replaced by two myristates. First, deacylation and remodeling occurs at the sn2 position and then, similarly at the sn1 position. Though the sn2 remodelase has been identified in T. brucei as TbGUP1, the sn1 remodelase is still unknown. Our goal was to identify the VSG GPI-anchor sn1 remodelase. Since GPI remodeling occurs inside the endoplasmic reticulum (ER), we hypothesized the second remodelase is an ER localized protein with acyltransferase activity. We screened the T. brucei genome database for candidates using the following criteria: homology to TbGUP1 and the other known remodelases, annotated acyltransferase domain homology, ER targeting signals, and transmembrane domains. We identified 16 candidate genes for the sn1 remodelase, six of them are annotated as acyltransferases in TriTryp database. We generated RNAi cell lines for two of the candidate genes and found that RNAi of the two candidates had no effect on growth in culture. This project will require future work to generate RNAi cell lines for the remaining candidates, to determining their localization by epitope tagging and fluorescence microscopy, and to assess their requirement for VSG GPI remodeling using in vitro remodeling assays and lipidomic analysis