Characterizing a novel endonuclease in Trypanosoma brucei

Shelley, Sam and Benson, Fiona and McKean, Paul (2021) Characterizing a novel endonuclease in Trypanosoma brucei. Masters thesis, Lancaster University.

Text (MSc Samuel Shelley 30 MAR 2020)
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Abstract

In 2012 the World Health Organisation published their Neglected Tropical Diseases Roadmap outlining 17 diseases which cause a significant burden across the world, namely in those areas stricken by poverty. The evidence outlined therein explained how with greater surveillance and management of these diseases, as well as increasing access to healthcare in the affect areas, should see these diseases brought under control, if not eradicated completely. One such disease is Human African Trypanosomiasis - or Sleeping Sickness – a parasitic disease caused by Trypanosoma brucei. Causing fevers joint pains and rashes at first, the disease progresses to cause a variety of neurological symptoms, before leading to a coma and eventually death. This parasite is able to evade the immune system by expressing an interchangeable coat of Variable Surface Glycoproteins coded by a vast library of different genes, allowing members of the parasite burden to escape immune detection long enough to be passed on to the next patient. To switch between these different genes, T. brucei relies on a series of different mechanisms, several of them underpinned by the process of homologous recombination. This report aims to explore the burden caused by this parasitic disease, the biology of the parasite itself and the mechanisms that underpin its pathogenicity. Furthermore, with previous studies having identified an enzyme with homology to the human FEN1 protein (an endonuclease responsible for cleaving the 5’ flaps generated during DNA replication and repair), this report aims to examine the structure and function of this putative TbGEN1 in homologous recombination and VSG-switching, and explore how this may help combat this disease. The research carried out for this report found that the putative TbGEN1 showed minimal Holliday junction cleavage activity but was far more effective at cleaving flap junctions. Through site-directed mutagenesis, a total of 9 substitution mutants were identified that lost all 5’-flap cleavage activity, with 7 specific residues (D34, D90, E164, D183, D185, G235, and D237) being implicated as key to TbFEN1’s activity. Furthermore, localisation studies carried out in vivo in procyclic trypanosomes would support this hypothesis, as the fluorescent tagged protein was not observed outside the nucleus, although this also raised further questions about the exact role of the target protein. The findings of this report demonstrate that there is still much we do not know about the fine workings of this parasite’s biology, but that with further research the possibility of finding a druggable target in T. brucei is very real.