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Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo

Awuah-Mensah, Georgina; McDonald, Jennifer; Steketee, Pieter C.; Autheman, Delphine; Whipple, Sarah; D'Archivio, Simon; Brandt, Cordelia; Clare, Simon; Harcourt, Katherine; Wright, Gavin J.; Morrison, Liam J.; Gadelha, Catarina; Wickstead, Bill

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Jennifer McDonald

Pieter C. Steketee

Delphine Autheman

Sarah Whipple

Simon D'Archivio

Cordelia Brandt

Simon Clare

Katherine Harcourt

Gavin J. Wright

Liam J. Morrison


Christine Clayton


Copyright: © 2021 Awuah-Mensah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection–enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite.


Awuah-Mensah, G., McDonald, J., Steketee, P. C., Autheman, D., Whipple, S., D'Archivio, S., …Wickstead, B. (2021). Reliable, scalable functional genetics in bloodstream-form Trypanosoma congolense in vitro and in vivo. PLoS Pathogens, 17(1), Article e1009224.

Journal Article Type Article
Acceptance Date Jan 17, 2021
Online Publication Date Jan 22, 2021
Publication Date Jan 22, 2021
Deposit Date Jan 24, 2021
Publicly Available Date Jan 29, 2021
Journal PLoS Pathogens
Print ISSN 1553-7366
Electronic ISSN 1553-7374
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 17
Issue 1
Article Number e1009224
Keywords Immunology; Genetics; Molecular Biology; Microbiology; Parasitology; Virology
Public URL
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