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Abstract

Trypanosoma parasites are the pathogenic agent causing human and animal African Trypanosomiasis. The parasites undergo a process called antigenic variation, which allows them to perpetually evade the host immune response. Trypanosomes are coated almost uniformly with a single protein, the Variant Surface Glycoprotein (VSG). This protein exists in over 1000 distinct alterations within the genetic repertoire of the parasite. These highly antigenic membrane proteins will trigger an extreme immune reaction, which are effective in clearing parasite. However, some cells in the pathogen population will switch to another VSG coat protein. This mechanism introduces a novel antigenic surface to the current Ig response, rendering it non-effective. The first two published protein structures lead to the assumption that all VSGs’ N-terminal architecture is highly conserved. However, these findings are deceptive. Recently published structures revealed that the N-terminus diverges far more than initially anticipated. Furthermore, it has been historically proposed that the VSG coat is not penetrable. High-density packing and the initial structural insight lead to the belief that only the upper portion of the N-terminal domain was likely to interact with the immune system. More recent reviews evaluated the data on this hypothesis and found that the evidence is lacking, suggesting that the coat is more accessible than previously claimed. This thesis aims to map binding epitopes on the VSG N-terminal domain, using high-resolution crystallographic data of VSG-nanobody complexes. Two epitopes could be verified, but these surprisingly did not occur on the upper surface of the VSG protein, but deeply buried. Three nanobodies were found to bind 50 Å below the top of protein at the three-helix bundle, and one other nanobody was found to bind the bottom lobe, 80 Å below the top surface. These findings suggest that, indeed, the antigenic surface coat of Trypanosoma parasites is not an inaccessible “shield”, but rather major parts of the N-terminal domain are exposed to the immune system. This data challenges the historical view on the parasite surface organisation and recasts the understanding of the host-pathogen interaction in this disease. Finally, in collaboration with the Engstler lab at the University Würzburg, we found that one of these nanobodies has a toxic effect on live parasites. Incubation with this nanobody induces high motility inhibition and eventually leads to death of the parasite. Electron micrographs revealed that the nanobody’s introduction results in the creation of excess membrane particles. These particles are likely the results of nanobody-induced membrane fission events. These events usually require a dedicated protein machinery paired with high energetic efforts, while the system described here in vivo is passively driven by protein-protein crowding on the dense VSG surface.