The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholars Program

GVSP Summer 2011 Scholars

Georgia Veterinary Scholar	Faculty Mentor

Andrew Durden UGA College of Veterinary Medicine Class of 2014	Dr. Roberto Docampo Department of Cellular Biology and The Center for Tropical and Emerging Global Diseases UGA Franklin College of Arts and Sciences

Localization and Functional Analyses of the Trypanosoma brucei Vacuolar Protein Sorting 41 (TbVPS-41)

Andrew C. Durden, Guozhong Huang, and Roberto Docampo

Trypanosoma brucei is a parasitic protist species that causes African trypanosomiasis (or sleeping sickness) in humans and nagana in animals in Africa. T. brucei possesses electron-dense acidic organelles rich in calcium and polyphosphate called acidocalcisomes. These organelles, which we first identified in trypanosomes, are conserved from bacteria to man. Previous work has demonstrated that acidocalcisome biogenesis depends on adaptor protein-3 (AP-3) complex (Huang et al., 2011). Adaptor protein complexes are important mediators for vesicular transport of membrane proteins between cellular compartments, such as Golgi complex, endosomes, lysosomes, and plasma membrane and usually interact with clathrin to transport cargo between compartments. AP-3 is mainly involved in sorting proteins to lysosome-related organelles in mammals and to acidocalcisomes in T. brucei although in trypanosomes it is unable to bind to clathrin. Only one other protein, with similar features to clathrin, has been characterized with selective function in AP-3 vesicle formation in yeast: Vps41p. Interestingly, knockdown of the Vps41 orthologue in T. brucei (TbVPS-41) resulted in an increase in the number of intracellular vesicles that resemble acidocalcisomes (Lu et al., 2007). The purpose of this study is to investigate where TbVPS-41 is localized in T. brucei and whether acidocalcisome biogenesis is affected by knockdown of TbVPS-41. With the use of C-terminal in situ PCR tagging, the targeted TbVPS-41 gene was tagged with an HA or Ty1 epitope (Oberholzer et al., 2006) and then transfected into the procyclic and bloodstream forms of the parasite. These transfected parasites underwent 1-2 week selection process. Western blot analysis was used to demonstrate gene expression and immunofluorescence assays were done to localize it using antibodies against the tags. Current experiments will examine the phenotypic changes occurring after knockdown of this gene by RNAi.

Huang, G., Fang, J., Sant'Anna, C., Li, Z-H, Wellems, D.L., Rohloff, P., and Docampo, R. (2011) Adaptor protein-3 (AP-3) complex mediates the biogenesis of acidocalcisomes and is essential for virulence in Trypanosoma brucei. Submitted.

Lu, S., Suzuki, T., Iizuka, N., Ohshima, S., Yabu, Y., Suzuki, M., Wen, L., and Hota, N. (2007). Trypanosoma brucei vacuolar protein sorting 41 (VPS41) is required for intracellular iron utilization and maintenance of normal cellular morphology. Parasitology 134, 1639-1647.

Oberholzer, M., Morand, S., Kunz, S., and Seebeck, T. (2006). A vector series for rapid PCR-mediated C-terminal in situ tagging of Trypanosoma brucei genes. Mol Biochem Parasit 145, 117-120.