Trypanosoma brucei is a hemoflagellate that causes African Sleeping Sickness in humans and nagana in cattle. The T. brucei surface is coated with ten million identical copies of single Variant Surface Glycoprotein (VSG), one of ~2500 VSGs encoded in the genome. VSG helps T. brucei evade host immune response by two mechanisms: antigenic variation (changing the surface coat) and endocytosis (internalizing immune complexes, recycling the VSG while degrading the immune components). For both strategies, 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. To reduce FAS, we use RNAi-mediated depletion of Acetyl-CoA Carboxylase (ACC), the first enzyme in the FAS pathway. We found that ACC RNAi resulted in a significant 30-40% reduction in both receptor-mediated and fluid-phase endocytosis. 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 resulted in ~40% decrease in surface VSG half-life (plt;0.05). Currently, we are examining two possible mechanisms leading to increased VSG turnover upon ACC RNAi: shedding in the media or intracellular degradation. Our preliminary data rules out degradation by proteasome or lysosome and reveals that ACC RNAi altered shedding of multiple surface proteins, including VSG. For the future, we will use proteomics to identify and quantify the proteins shed in the media, and we will investigate the mechanisms of their release.
