Abstract
Summary Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising ∼10% total protein. The active VSG gene is in a Pol I‐transcribed telomeric expression site (ES). We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei. Mutational analysis of the ectopic VSG 3′UTR demonstrated the essentiality of a conserved 16‐mer for mRNA stability. Expressing ectopic VSG117 from different genomic locations showed that functional VSG levels could be produced from a gene 60 kb upstream of its normal telomeric location. High, but very heterogeneous levels of VSG117 were obtained from the Pol I‐transcribed rDNA. Blocking VSG synthesis normally triggers a precise precytokinesis cell‐cycle checkpoint. VSG117 expression from the rDNA was not adequate for functional complementation, and the stalled cells arrested prior to cytokinesis. However, VSG levels were not consistently low enough to trigger a characteristic ‘VSG synthesis block’ cell‐cycle checkpoint, as some cells reinitiated S phase. This demonstrates the essentiality of a Pol I‐transcribed ES, as well as conserved VSG 3′UTR 16‐mer sequences for the generation of functional levels of VSG expression in bloodstream form T. brucei.
Highlights
The African trypanosome Trypanosoma brucei is a paradigm for monoallelic control and antigenic variation
Trypanosoma brucei faces relentless immune attack in the mammalian bloodstream, where it is protected by an essential coat of Variant Surface Glycoprotein (VSG) comprising 10% total protein
We investigated factors mediating these extremely high levels of VSG expression by inserting ectopic VSG117 into VSG221 expressing T. brucei
Summary
The African trypanosome Trypanosoma brucei is a paradigm for monoallelic control and antigenic variation. T. brucei is the causative agent of Human African Trypanosomiasis and ‘nagana’ in livestock, which are transmitted by tsetse flies. Case numbers for trypanosomiasis have been falling, 70 million people are still estimated to be at potential risk of infection (Franco et al, 2014). In addition to the human mortality, livestock diseases caused by T. brucei and related trypanosomatids cause enormous economic losses. A chronic infection is maintained through a highly sophisticated strategy of antigenic variation, based on the monoallelic expression of Variant Surface Glycoprotein (VSG) (Glover et al, 2013b; Gunzl et al, 2015; Duraisingh and Horn, 2016)
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