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Abstract

Trypanosoma brucei (T. brucei) is an extracellular unicellular pathogen, which is the causative agent of African sleeping sickness in sub-Saharan Africa. It has a two-host life cycle in its vector tsetse fly (Glossina sp.) and its mammalian hosts. The work of this thesis focuses on the metacyclic stage of the parasite, which is the mammalian infectious form injected by the fly, and the metacyclic Variant Surface Glycoprotein (mVSG). VSG is expressed in the metacyclic and bloodstream forms. The latter differentiate from metacyclic in the mammalian host and are capable of evading the host immune system by constantly changing the expression of its surface proteome makeup. Despite a vast number of VSG genes present in the genome repertoire (~2000 genes and pseudogenes), only a specific subset of five VSGs, called metacyclic VSGs (mVSGs), are expressed in the metacyclic stage of the Lister 427 strain. In this study, we are comparing mVSGs and bloodstream VSGs at the protein structure level. Using structure-based prediction by protein threading, we showed that VSGs in T. brucei are divided into two different structure classes, A and B. We have determined the structure of mVSG 531 and mVSG1954, classified in Class A and B, respectively, by X-ray crystallography. The homodimer structure of mVSG531's Nterminal domain shows the conservation of the three-helix bundle core and the dumbbell-like shape observed in the previously determined VSGs structures in Class A, VSG2 (MITat 1.2) and VSG1 (MITat 1.1), which are expressed in the bloodstream form. The structure of mVSG1954 also shows similarities to VSG3 (MITat 1.3), classified in Class B and expressed in the bloodstream form. Like VSG3, it exists as a monomer in the crystal asymmetric unit, but also and forms non-crystallographic trimers. While the VSG3 is O-glycosylated, there is no evidence of O-glycosylation in the mVSG1954 structure. The results address a long-standing question of whether the mVSGs were similar to or different from the bloodstream VSG coat proteins, showing that structurally and biochemically, the two life-stages classes of VSGs are very similar.