Abstract
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.
Highlights
A critical tool for understanding pathogen biology is the ability to make genetic modifications, especially creating specific mutants of target genes that can be used to investigate the locations of gene products, the effects of changes in expression, or consequence of complete gene removal
Work on African trypanosomiasis (AAT) is severely limited by difficulties in making even small genetic modifications and lack of tools for many functional genetics applications
Animal African trypanosomiasis (AAT) is a parasitic disease associated with anaemia, loss of condition and death in sub-Saharan livestock
Summary
Animal African trypanosomiasis (AAT) is a parasitic disease associated with anaemia, loss of condition and death in sub-Saharan livestock. The disease in cattle is caused by trypanosomes of the species Trypanosoma brucei (sub-species of which cause human disease), T. vivax, and T. congolense Of these species, T. congolense may cause the majority of disease in sub-Saharan cattle [2,3], as well as making up a substantial proportion of infections [4]. There are substantial differences in the differential regulation of genes involved in specific biological processes during infection [12] and between metabolomes (Steketee et al, in preparation). Such differences create a pressing need for species-specific models of AAT that can be used and modified in axenic culture and transmitted through animals
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