Authors

M Soale²; P Steketee³; R Young³; F Giordani¹; M Barrett¹; L Morrison⁴; B Wickstead²; C Gadelha²;  ¹ Institute of Infection, Immunity and Inflammation, University of Glasgow, UK;  ² University of Nottingham, UK;  ³ The Roslin Institute, University of Edinburgh, UK;  ⁴ Roslin Institute, The University of Edinburgh, UK

Discussion

Trypanosoma vivax is the most widespread causative agent of animal African trypanosomiasis (AAT). This parasite accounts for more than 50% of cattle infections in endemic areas in West Africa and all AAT in South America. Despite its huge veterinary importance, it is the least studied among the three major AAT species (T. brucei, T. congolense and T. vivax). As a result, key parts of T. vivax biology are poorly understood, including aspects of metabolism, transmission, pathology, antigenic variation and drug sensitivity. Here, we report the development and application of a toolkit for regulatable and scalable functional gene studies using in vitro-derived T. vivax trypomastigotes. Through development of a specifically tailored cell line expressing T7 RNA polymerase and Tet repressor protein, we show silencing of both essential and non-essential genes using a species-specific RNA-interference tool. We further demonstrated conditional knockouts of essential genes and genes associated with drug resistance – including using a highly efficient Cas9-mediated approach – leading to the identification of genetic determinants of resistance to two trypanocides. Moreover, by modifying a specific unique locus with a self-excising gene encoding I-SceI nuclease, we developed a new cell line that can produce >10,000 independent clones in a single transfection, enabling the generation of high-complexity mutant libraries. In addition to this work providing a platform for understanding drug resistance in T. vivax, we are using it to explore the molecular mechanisms underlying T. vivax antigenic variation and the biology of its bloodstream form.