Awuah-Mensah, Georgina
(2021)
Exploiting trypanosomes as models for pathogenicity and core biology research.
PhD thesis, University of Nottingham.
Abstract
Model systems are commonly used to investigate biological processes and understand molecular mechanisms. The choice of a model cell or organism is informed by the scope of the question being asked. Decades of research using predominantly animal and fungal models form the bulk of our knowledge of eukaryotic processes. The diversity of eukaryotic organisms, however, indicates that this may not be representative of what exists in more diverged groups, and key processes like cell division vary across the larger group. In the past, non-traditional models including Tetrahymena have been applied to answer biological questions because of peculiar traits that make them suitable for investigation. African trypanosomes offer an excellent opportunity as model organisms because of their divergence from common models and importance to human health and livelihood. Particularly Trypanosoma brucei, with several genetic tools available to study them, has become an interesting model to study processes like cell division, cytoskeleton and antigenic variation. Here, I present work exploring the application of two species of African trypanosomes as models to investigate common biology of eukaryotic kinesins and specific biology of animal African trypanosomiasis.
The genetic tractability of T. brucei and its cytoskeletal structure, which is predominantly made up of tubulin, makes it a good model to study functional differentiation between kinesin families. I show cellular localisation and characterise the effect of RNAi depletion of representative kinesin families. The data suggest that Kinesin-2, -3 and 13-1 perform similar roles in T. brucei as seen in other systems, and can therefore be candidates for investigation. On the contrary, loss of both alleles of T. brucei Kinesin-1 does not affect in vitro growth, suggesting possible functional redundancy in trypanosomes.
Trypanosoma congolense is the most significant cause of animal African trypanosomiasis, a wasting disease of livestock that results in huge economic losses to agriculture particularly in Africa. Disease presentation differs from T. brucei, and the lack of genetic tools makes it difficult to study the biology of T. congolense, specifically its pathogenesis in animal trypanosomiasis. I show the progressive development and application of genetic tools for endogenous locus tagging, RNA interference and high-complexity mutant library production. I demonstrate that loci on the minichromosome can be targeted for transgene integration, give good expression and are regulatable. Genes targeted by RNAi could only be depleted to about 50% of original levels, with similar proportion of cells expressing mutant phenotype, suggesting that T. congolense has lower RNAi penetrance than T. brucei. The development and optimization of a system that uses a homing endonuclease to increase DNA breaks at integration sites consistently increased the number of positive transformants to ~ 50,000 per transfection. This T. congolense line maintained stable transfection efficiency even after cycles of freeze-thawing.
Finally, I demonstrate for the first time, a genome-wide technology in T. congolense. Using all the tools developed, I create two RNAi libraries containing > 5x105 independent transformants. The method of library generation by direct RNAi fragment sequencing (DRiF-Seq) allowed detection of very small changes in gene fitness cost due to RNAi. This enabled fitness screen of > 9000 genes in T. congolense even at a low RNAi penetrance. Analysis of differential biology between T. congolense and T. brucei gives some glimpse into similarities and differences in processes like endocytosis and quorum sensing. I also demonstrate application of the T. congolense RNAi libraries in a drug screen to investigate isometamidium mode of action and resistance.
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