MORN repeat proteins and the Trypanosoma brucei bilobe

The protist parasite Trypanosoma brucei, causative agent of African sleeping sickness in man and Nagana in cattle, is an early-branching Eukaryote of fundamental biological interest. We have identified a novel cytoskeletal structure in this organism with a bilobed morphology demarcated by TbCentrins 2 and 4. Centrins are small proteins belonging to the EF-hand superfamily and are highly conserved components of the microtubule organizing centers of Eukaryotic cells. This proposal focuses on a novel component of the bilobe, TbMORN1, which we recently discovered. TbMORN1 is composed of 15 tandem 23-amino acid MORN repeats, which are found throughout the tree of life but whose function is still unclear. It is thought that they are used as protein-protein or protein-lipid interaction domains. TbMORN1 is localized exclusively to the bilobe structure, unlike the TbCentrins which are also present at the basal body and other cytoskeletal architectures. TbMORN1 is also essential for the viability of the bloodstream form of the parasite. Using targeted depletion by RNAi we intend to gain a greater understanding of the biological function of TbMORN1. We also aim to identify other bilobe components by identifying its intracellular binding partners, and gain insight into its mechanism of operation through high resolution structural studies.

This project has led to a major advance in our understanding of a human parasite. The parasite in question is Trypanosoma brucei, a single-celled organism that causes the neglected tropical disease human African trypanosomiasis (HAT, colloquially known as Sleeping Sickness). T. brucei has a two-stage life cycle, alternating between the tsetse fly and mammalian hosts. There is an urgent need for new drugs against T. brucei owing to limited number of options and high toxicity. One of the problems with finding new drug targets against T. brucei is that it is a eukaryote (meaning that its genetic material is enclosed within a nucleus). Humans are also eukaryotes, so it is much more difficult to find a drug which kills T. brucei that is not also toxic to us. This is very different from the situation with prokaryotes (bacteria), which are sufficiently different that antibiotics can be safely used against them.Like all eukaryotic cells, T. brucei has an internal architecture - the cytoskeleton. Numerous studies have shown that the cytoskeleton of T. brucei is essential to its viability and pathogenicity. The work undertaken in this project focused on a newly-discovered component of the T. brucei cytoskeleton called the bilobe. When the project began, only one protein was known to be exclusively present at the bilobe - TbMORN1.By specifically depleting TbMORN1 from cells, it was shown that the bilobe is essential for the viability of the mammalian-infective form of T. brucei. A combination of fluorescence microscopy and electron microscopy was used to study the bilobe at high resolution, which led to a new model for its morphology. By applying a new technique called proximity-dependent biotin identification (BioID), a total of seven new bilobe proteins were identified in T. brucei. This also allowed the delineation of a number of subdomains within the bilobe structure. Finally, the TbMORN1 protein was studied in vitro in order to generate an atomic-resolution structural model using several biochemical and biophysical methods.