The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in the tsetse fly salivary glands

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.
Author summary African trypanosomes are parasitic protists which cause endemic disease in sub-Saharan Africa. To evade mammalian immune responses the parasite has developed a system of antigenic variation, where the surface of the cell is covered in a tightly packed coat of variant surface glycoproteins (VSGs). Each cell expresses only one variant surface glycoprotein at a time, and this is periodically switched to evade new antibodies. The process of singular gene expression is termed monoallelic expression and this has two components, establishment and maintenance, i.e. how a single gene is selected for expression and how its singular expression is maintained throughout successive generations. The establishment of monoallelic VSG gene expression occurs in the salivary gland of the tsetse fly vector, although this process is not well understood. We used single cell gene expression profiling applied to thousands of single cells in the salivary gland of the fly. We show that in order to select a single gene, trypanosomes initially transcribe multiple VSGs before a single gene is selected for high-level expression. We propose a model where this process is driven by a race to accumulate transcription factors at a single VSG gene.