Expression and behaviour of GPI-anchored surface proteins in trypanosomes
There are hundreds of species in the genus Trypanosoma that proliferate in the bloodstream and tissue spaces of vertebrate hosts. African trypanosome species have evolved a variant surface glycoprotein (VSG) coat which underpins a population survival strategy based on antigenic variation. However, the vast majority of trypanosome species do not use this strategy and our understanding of the immune evasion strategies used by these species is far less developed. In some species including Trypanosoma theileri, it has been shown that the surface comprises of a mixture of a large number of different proteins, some of which are GPI-anchored. This thesis explores the expression and behaviour of trypanosome surface proteins and is split into two parts. The first part focuses on the link between the expression of VSG and the control of cellular growth in the African trypanosome species Trypanosoma brucei. The second part explores the behaviour of surface proteins in the non-African trypanosome species Trypanosoma theileri and Trypanosoma carassii. The VSG of T. brucei is GPI-anchored and is monoallelically expressed from a repertoire of hundreds of genes. The active VSG gene is located within a specialised telomeric expression site and is transcribed by RNA polymerase I. VSG is an essential protein, knockdown of the active VSG mRNA using RNAi results in a pre-cytokinesis cell cycle arrest, resulting from a threshold requirement of VSG for cellular proliferation. It is not known how the cell monitors the integrity of the VSG coat, nor how this information is relayed to the cell cycle control mechanisms. An assay was developed to determine whether an alternative GPI-anchored protein could substitute for a VSG in maintaining cell proliferation. The assay used a morpholino to knockdown VSG translation, and alone this is sufficient to produce the phenotype observed following VSG knockdown using RNAi. Since the morpholino prevents translation but leaves the VSG mRNA intact, it can be concluded that the presence of VSG mRNA is not sufficient to prevent cell cycle arrest. The cell cycle arrest could be rescued by inducing the expression of a second VSG from a T7 promoter. This observation proves that expression of VSG from an expression site is not essential for progression of the cell cycle. VSG mutants, fluorescent proteins and other trypanosome surface proteins were tested for their ability to rescue growth. Only VSG with a truncated linker region between the C and N-terminal domains could replace the naturally expressed VSG. It can be concluded that the formation and functionality of VSG proteins is carefully monitored and communicated to the controls of cell division. Several African trypanosome species, including T. brucei, are known to passively sort antigen-immunoglobulin complexes, resulting from hydrodynamic flow, to the posterior end of the cell, effectively concentrating them in the flagellar pocket where they are endocytosed. Here, the question was asked: was this self-cleaning process present in the ancestor of African trypanosomes or has it evolved subsequent to the VSG coat? The ability of two trypanosome species, Trypanosoma theileri and Trypanosoma carassii to remove surface bound biotin-streptavidin and biotin-IgG complexes from their surfaces was tested. T. theileri is an extracellular Stercorarian parasite of cattle and is in a sister group to African trypanosomes, whereas T. carassii infects carp and diverged before the split between the Stercorarian and Salivarian trypanosomes. I show that neither the accumulation of surface bound IgG towards the posterior pole nor rapid endocytosis occurs in either species. This suggests that the IgG does not protrude sufficiently to be subjected to hydrodynamic flow forces and/or the GPI-anchored cell surface proteins do not have the same ability to diffuse over the cell surface as VSG. The observations also suggest that the VSG coat has allowed the African trypanosomes to evolve a self-cleaning surface in addition to its role in antigenic variation.
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