Methods for studying the cell cycle in African Trypanosomes

Pendlebury, Max and Urbaniak, Mick (2024) Methods for studying the cell cycle in African Trypanosomes. Masters thesis, Lancaster University.

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Abstract

African trypanosomes are a genus of unicellular parasitic organisms that cause both human and animal African trypanosomiasis. While HAT, caused by Trypanosoma brucei rhodesiense or gambiense, has shown record low cases in recent years, the elimination of AAT remains difficult due to many complicating factors. One of these factors is the comparatively poor understanding of the main causative agent of AAT, Trypanosoma congolense. Both T. brucei and T. congolense possess a single flagellum that originates from inside the cell and exits through a membrane invagination called the flagellar pocket. The flagellar pocket and its associated structures are not only critical for parasite survival and virulence but also for coordinating the cell cycle. Improving our understanding of cell cycle regulation in these species could lead to novel drug targets, which could be aided by utilisation of a suitable synchronisation method. This project aims to optimise protocols for both the synchronisation and enrichment of flagellar pocket associated cytoskeletal structures (F-PACS) in African trypanosomes to provide opportunities for greater analysis of the cell cycle. To synchronise T. brucei, centrifugal counter-flow elutriation (CCE) was used to separate cells based on their hydrodynamic volume. Enriched fractions were collected containing >97% of cells in G1 in PCF, and >98% in BSF. Once returned to culture, this population continued to grow in a synchronous fashion. A protocol for enrichment of F-PACS in PCF T. brucei was optimised. Synchronised samples were generated for analysis of changes in phosphosite abundance across the cell cycle. Attempts were made to optimize mechanical and chemical synchronisation techniques for use on T. congolense, achieving a ~76% enrichment in G1 by CCE. This project provides useful optimisations for continued study of the cell cycle in trypanosomes.