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

Abstract

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.

Single-cell transcriptomics reveals expression profiles of Trypanosoma brucei sexual stages

“…Several BARP genes were the prominent marker transcripts in cluster C4 (Figure 1D), identifying this cluster as salivary gland epimastigotes, consistent with previous studies (Vigneron et al, 2020;Hutchinson et al 2021). The majority of the later time point (day 40 pi) cells were found in clusters C5 and C6 (Figure 1B, C), showing that these clusters represent the later salivary gland developmental stages including mature metacyclics and/or their immediate precursors, pre-and nascent metacyclics.…”

“…Multiple mVSGs were also expressed by 38% (6/16) and 21% (4/19) cells in clusters C4 and C6 respectively, and a single cell in C3 (Figure 2C, 2D). Hutchinson et al, 2021 confirmed expression of two mVSGs in premetacyclics using single molecule mRNA-FISH and put forward a model where multiple mVSGs are transcribed at low levels initially, with a single mVSG dominating expression in the mature metacyclic forms. Based on this model, C4, C5 and C6 all contain a high proportion of pre-metacyclics, as well as some mature metacyclics.…”

“…Mature metacyclics can be unequivocally identified by their lack of expression of the cell surface proteins GPEET, EP and BARP, replaced by expression of a single mVSG gene in each cell (Christiano et al, 2017), but pre-metacyclics also transcribe mVSGs at high level in (Hutchinson et al, 2021). To explore mVSG expression in salivary gland-derived cells here, we first needed to identify the mVSG repertoires of strains 1738 and J10, which differ from those of previously published strains.…”

“…Single-cell RNAseq opens the door to study heterogenous populations of single-celled parasites by delineating expression patterns of individual cells allowing us to understand continuous developmental processes, cell-type specific patterns of co-expression and bethedging strategies (Howick et al, 2019;Nötzel and Kafsack, 2021;Poran et al, 2017;Real et al, 2021;Reid et al, 2018;Sà et al, 2020;Witmer et al, 2021). Recent studies have profiled T. brucei populations using single-cell droplet-based approaches from BSF culture to profile the development of stumpy forms (Briggs et al, 2021) as well as in vivo salivary gland parasites in order to identify a potential vaccine candidate among mature metacyclics (Vigneron et al, 2020) and the dynamics of VSG expression in the developing metacyclic parasites (Hutchinson et al, 2021). These studies complement the previous bulk transcriptomic studies in T. brucei that identified the major changes in transcriptional patterns over time and metacyclic development using either whole infected salivary glands or in vitroderived metacyclics from the RBP 6-inducible system (Christiano et al, 2017;Doleželová et al, 2020;Kolev et al, 2012;Naguleswaran et al, 2018;Ramey-Butler et al, 2015;Savage et al, 2016Savage et al, , 2012Telleria et al, 2014).…”

May the Odds Be Ever in Your Favor: Non-deterministic Mechanisms Diversifying Cell Surface Molecule Expression

“…VSG expression initiates in parasites that reside in the salivary gland of the tsetse fly, prior to bloodstream infection. Recent data has shown that multiple VSG genes are initially transcribed within pre-metacyclic cells, with a single gene being expressed within mature metacyclic cells ( Hutchinson et al, 2021 ). A “race” model has been proposed to explain this phenomenon in which different VSG expression sites race to hit a certain threshold level of transcription.…”