Authors

F Taha²; MC Taylor¹; MA Child²; C Tiengwe²;  ¹ London School of Hygiene and Tropical Medicine, UK;  ² Dept. Life Sciences, Imperial College, University of London, UK

Discussion

African trypanosomiasis, caused by various Trypanosoma brucei spp., affects both humans and animals in sub-Saharan Africa, and remains an impediment to socioeconomic development. During an infection, T. brucei exhibits tissue tropism with proliferative forms predominantly colonising specific tissue niches including the skin, adipose fat, and the brain, while transmissible forms are typically found in blood. The interactions between these different populations in the host and the bottlenecks they encounter are not well-understood. We are using barcoded T. brucei to quantitatively analyse the migration patterns and dynamics of these parasite populations to understand critical features of parasite virulence and disease pathology.
Using a versatile CRISPR-Cas9 method, we integrate sequence barcodes at the haploid AAT6 gene locus. We have successfully applied this technique to wild-type isogenic Lister 427 and bioluminescent EATRO1125 Antat 1.1 T. brucei strains, generating barcoded strain libraries containing 96 unique barcodes in single transfections. The frequency and proportion of each barcode (i.e. population structure) is stable in vitro for at least 30 days, and unaffected by cryo-preservation, indicating that barcoded parasites have equal fitness. This stable, neutral nature of the barcodes indicates that our system is robust and reliable for long-term lineage tracing and the analysis of parasite population dynamics in different stages of infection and host ecosystems. We are currently working on increasing the barcode library size and adapting our barcoding approach to insect-stage parasites. This will allow us to interrogate parasite distributions, tissue niche colonisation and genetic selection bottlenecks that shape the population structure at high resolution in both the vector and animal model.