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
