VSG expression in African trypanosomes

: Gene silencing, cell cycle and developmental control

Abstract

African trypanosomes are major human and animal pathogens in sub-Saharan Africa that cycle between a mammal and tsetse fly vector. In the mammalian host, these parasites exist exclusively extracellularly. To evade the adaptive immune system, trypanosomes undergo antigenic variation of the variant surface glycoprotein (VSG) coat. The trypanosome genome contains ~2,000 VSG genes, however only a single VSG gene is transcribed with several expression site associated genes (ESAGs) in a polycistronic transcription unit by RNA-polymerase I, from one of approximately 15 telomeric VSG expression site (VSG-ES). This produces super-abundant transcript and protein; approximately 10 % of the cells gene expression is dedicated to this single gene. In the tsetse fly, VSG expression is switched off, and the cell surface remodeled. I have investigated the mechanisms by which the trypanosome cell maintains monoallelic expression of VSG genes. Firstly, I have characterised the novel protein VEX1, which was identified in an RNAi library screen for regulators of VSG silencing. VEX1 controls the monoallelic expression required for antigenic variation. RNA-seq analysis shows that VEX1 mediates both positive and negative control of VSG-ES. A novel form of RNA mediated silencing mediates repression of the remaining VSG-ES; both VSG and TERRA transcripts are implicated in silencing. Next, as telomeric expression sites are highly repetitive; I develop tools to demonstrate the feasibility of applying next generation sequencing technologies to these loci. Analysis of high confidence isogenic RNA-seq read alignments from mammalian and insect life cycle stages revealed differential control of ESAG10 and the tsetse specific metacyclic VSG genes. Further, analysis of VEX1 perturbed transcriptomes shows that VEX1 mediated silencing affects both the telomere proximal VSG gene and promoter proximal ESAGs. I next characterised effect of inhibiting VSG translation on the cell cycle with the MS2 coat protein. This revealed distinct roles for the VSG transcript and protein in cell cycle regulation. In addition, these data explain the developmentally regulated uncoupling of DNA replication and cytokinesis observed exclusively in the bloodstream trypanosome. Finally, I conduct an RNAi library screen in insect stage cells for factors controlling developmental regulation of VSG promoters, highlighting a link to carbohydrate sensing.

Date of Award	2016
Original language	English
Supervisor	David Horn (Supervisor)