McDonald, Jennifer
(2018)
Probing the system behind variable antigen expression in animal pathogen Trypanosoma congolense.
PhD thesis, University of Nottingham.
Abstract
Antigenic variation is a strategy adopted by many pathogens to evade the host immune system. African trypanosomes, in particular Trypanosoma brucei, are models for antigenic variation in parasites. These organisms periodically switch the identity of their major surface protein, variable surface glycoprotein (VSG), and as a result are able to survive extracellularly in the blood of vertebrate hosts for extended periods. In T. brucei, there are over 3000 VSG genes but only 1 is transcribed at a time from a telomeric expression site. Apparently uniquely, RNA Polymerase I (Pol I) transcribes the active expression site and this occurs in an extranucleolar location, known as the expression site body.
Trypanosoma congolense is the most significant causative agent of Animal African Trypanosomiasis, a wasting disease of livestock that is a severe burden to agricultural productivity in Africa. T. congolense has VSG-like proteins that are also on the cell surface and undergo antigenic variation. However, little is known about the molecular basis for this variation. The T. congolense genome has been sequenced but the chromosome ends are not assembled, making the existence and structure of any subtelomeric expression sites unclear. In addition, data suggest that surface proteins, potentially including VSG, may be transcribed by RNA Polymerase II.
To investigate the structure of potential expression sites in T. congolense, I used Transformation Associated Recombination cloning to capture chromosome ends as yeast artificial chromosomes. Ends could be captured with 3 independent DNA baits predicted to be subtelomeric. Sequencing of a subset of these confirmed the presence of subtelomeric gene family members, but did not uncover likely VSG expression sites. A bioinformatic approach using hidden Markov models of Pol I promoters also failed to identify likely expression sites in the assembled sequence, but analysis suggested that DNA divergence between currently available species is too great for this analysis to be unambiguous.
As an alternative strategy, I generated insect-form (trypomastigote and epimastigote) and bloodstream-form cell lines stably expressing a fluorescent Pol I subunit. This showed an additional spot containing Pol I, in addition to the nucleolus, that is present only in the bloodstream form stage. This focus did not contain typical markers for the nucleolus and the polymerase contained was found to be active and resistant to α-amanitin. ChIP-seq identified genes associated with Pol I transcription, and also only a weakly enriched VSG that was shown by massspectrometry to be the main protein comprising the surface coat. As the VSG coat is the main surface protein in bloodstream form stage, it is expected to be expressed in abundance. This indicates there may be more than one VSG being expressed or the true VSG being expressed does not have sequence data available to be identified as such. These data strongly indicate that T. congolense VSG are transcribed by Pol I in the ESB, but from an expression site with a structure very different from T. brucei.
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