The goal of this proposal is to evaluate the endogenous secretory machinery responsible for the proper localization of virulence factors in the protozoan parasite Trypanosoma brucei. T. brucei causes African Human African trypanosomiasis (HAT), endemic to 36 countries of sub-Saharan Africa and responsible for tens of thousands of deaths annually. The disease is uniformly fatal without treatment, available treatment regimens are highly toxic and difficult to administer, and no vaccines exist. T. brucei i transmitted to the host bloodstream via the bite of an infected tsetse fly, and the success of the parasite during infection relies on two essential virulence components that are both products of the T. brucei secretory system. The first is the surface- localized glycosylphosphatidylinositol (GPI)-anchored Variant Surface Glycoprotein (VSG) coat. The second is the parasite lysosome, a digestive organelle that processes endocytosed nutritional factors and degrades lytic host immune complexes. The proper targeting of VSG to the cell surface and organellar constituents to the lysosome is central to the pathogenesis of T. brucei. Despite its crucial role during infection, the T. brucei secretory and endomembrane trafficking machinery remain widely uncharacterized. The first trafficking step encountered by all secretory cargo (independent of their final destinations) is exit from the ER in COPII (coat protein II)-coated vesicles. In other eukaryotes, members of the p24 family of transmembrane proteins form poorly characterized heteromeric complexes that mediate cargo selection during COPII vesicle budding. In particular, the two well-defined p24 complexes described (yeast &mammals) bind and select mature GPI-anchored proteins for ER exit. Bioinformatic analyses identified 8 members of the p24 family in the T. brucei genome (TbERP1-8), and we initially hypothesized that they might be involved in GPI- VSG trafficking. TbERP1-8 were evaluated individually via RNAi silencing. None appear to be essential, and only TbERP2 silencing caused a moderate growth defect. Additionally, the TbERP1-8 silenced cell lines exhibited no delays in VSG trafficking or in bulk secretion. However, silencing of TbERP1, TbERP2 or TbERP8 was sufficient to cause dramatic pre-Golgi delays in the forward trafficking of two endogenous lysosomal proteins, p67 and TbCatL. Additionally, silencing of TbERP1, TbERP2 or TbERP8 caused increased secretion of an ER resident protein, suggestive of a secondary regulatory role in protein targeting. The parallel phenotypes observed in these knockdown experiments suggest that Erp1, Erp2, and/or Erp8 function during ER exit and may comprise a p24 complex with novel cargo specificity(s). It is the goal of this proposal to fully characterize these candidates, and to describe the subunit identity and cargo specificity of the functional TbErp complex in T. brucei.

Public Health Relevance

Trypanosoma brucei causes Human African Trypanosomiasis, endemic in sub-Saharan African and the cause of tens of thousands of deaths annually. Current treatments are highly toxic and difficult to administer, and no vaccines exit, so a better understanding of the biology of this parasite may reveal potential avenues for intervention strategies. The virulence of T. brucei relies on two critical components, both produced by the parasite secretory system: 1) the Variant Surface Glycoprotein (VSG) coat, and 2) the parasite lysosome, a digestive organelle that processes endocytosed nutritional components as well as lytic host immune complexes. We propose a full characterization of the p24 family of transmembrane proteins in T. brucei, which are likely components of the endogenous early secretory machinery that traffics critical cargoes to the cell surface (VSG) and to the lysosome.

Agency
National Institute of Health (NIH)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32AI112254-01
Application #
8717799
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcgugan, Glen C
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14260