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Title: Investigating the linkage between Trypanosoma brucei pleomorphism and antigen switch frequency
Author: McWilliam, Kirsty Ross
ISNI:       0000 0004 7969 3072
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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African trypanosome infections are characterised by antigenic variation to avoid host immunity and by the production of transmission stages to promote disease spread. Laboratory-adapted ('monomorphic') lines of Trypanosoma brucei are reported to switch their expressed VSG antigenic coat at a much lower frequency than 'pleomorphic' populations recently transmitted through tsetse flies. These laboratory-adapted parasites also lose the capacity to differentiate into transmission competent 'stumpy forms'. It is unclear if the reduced rate of antigenic variation is directly coupled to the loss of pleomorphism or whether the processes, although co-selected by multiple passage, are independent. To address whether monomorphism caused a concomitant change in the frequency of antigenic variation, an 'inducible monomorphism' model was exploited. This exploited pleomorphic RNAi cell lines that would inducibly silence genes required for stumpy formation. This provided a tool to switch pleomorphism 'on' or 'off' inducibly, without long term passage. Thereafter, two approaches were used to ask if the induction of monomorphism directly influenced antigenic variation: in vitro flow cytometry-based VSG switch assays, and VSGseq, a targeted sequencing approach. These assays demonstrated that the induction of monomorphism did not reduce VSG switch rate nor generate a reduction in expressed VSG diversity, or change the expressed VSG subset. To extend this analysis further, the prolonged in vitro passage of a pleomorphic cell line was used to select isogenic monomorphic populations with reduced capacity to generate stumpy forms. These selected monomorphs cells did not exhibit a reduction in VSG switch rate compared to the parental pleomorphic population, thus corroborating the observations with the induced monomorphic cells. To understand the loss of pleomorphism in the selected cells, an 'evolve and resequence' approach and RNAseq analysis was adopted. Interestingly, the 'selected monomorphs' were depleted of transcripts whose expression was associated with the stumpy and insect forms of the parasite. Particularly, a number of CCCH zinc finger proteins, such as ZC3H20, were significantly downregulated. These RNA-binding proteins could represent novel regulators of slender to stumpy differentiation. Overall our results demonstrate that T. brucei antigen switch frequency and pleomorphism can be uncoupled, and provide new insight into the molecular control of stumpy formation.
Supervisor: Matthews, Keith ; Morrison, Liam Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Trypanosoma brucei ; Variable Surface Glycoprotein ; VSG surface coats ; VSG switch frequency ; differentiation capacity ; ZC3H20