Abstract
BackgroundAfrican trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes. The expressed VSGs are located at the ends of telomeric, polycistronic transcription units known as VSG expression sites (VSG-ESs). Each cell has many VSG-ESs but only one is transcribed in bloodstream-form parasites and all of them are inactive upon transmission to the insect vector mid-gut; a subset of monocistronic metacyclic VSG-ESs are then activated in the insect salivary gland. Deep-sequence analyses have been informative but assigning sequences to individual VSG-ESs has been challenging because they each contain closely related expression-site associated genes, or ESAGs, thought to contribute to virulence.ResultsWe utilised ART, an in silico short read simulator to demonstrate the feasibility of accurately aligning reads to VSG-ESs. Then, using high-resolution transcriptomes from isogenic bloodstream and insect-stage Lister 427 Trypanosoma brucei, we uncover increased abundance in the insect mid-gut stage of mRNAs from metacyclic VSG-ESs and of mRNAs from the unusual ESAG, ESAG10. Further, we show that the silencing associated with allelic exclusion involves repression focussed at the ends of the VSG-ESs. We also use the approach to report relative fitness costs following ESAG RNAi from a genome-scale screen.ConclusionsBy assigning sequences to individual VSG-ESs we provide new insights into VSG-ES transcription control, allelic exclusion and impacts on fitness. Thus, deeper insights into the expression and function of regulated multi-gene families are more accessible than previously anticipated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3154-8) contains supplementary material, which is available to authorized users.
Highlights
African trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes
Non sub-telomeric portion of the Trypanosomiasis research Edinburgh University (TREU)-927 reference and Lister 427 genomes are closely related, sequence reads were aligned to the 11 megabase chromosomes from the TREU-927 genome [29], plus a non-redundant set of the 14 VSG expression site (VSG-ES) [22] and 5 metacyclic VSG-ESs [15] from the Lister 427 strain
The analysis indicated that Bowtie2 aligns 75.5 % of in silico generated reads to the correct bloodstream VSG-ES with a MapQ > = 0 (98.7 % to metacyclic VSGESs) such that mis-aligned reads, as expected, can have a significant negative impact on transcriptome analysis
Summary
African trypanosomes cause lethal diseases in humans and animals and escape host immune attack by switching the expression of Variant Surface Glycoprotein (VSG) genes. The trypanosome surface is coated in a dense layer of 107 copies of a single variant surface glycoprotein (VSG) [2] Switching of this VSG coat is central to adaptive immune evasion, and operates at a rate of approximately 10−6 per parasite cell division in culture [3]. In vivo, this leads to the recrudescent parasitaemia characteristic of T. brucei infection [1], where unswitched parasites are removed by antibody mediated killing
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