The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq of Trypanosoma brucei in 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.

Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq

“…This suggests that cell fusion might be a response to DNA stress that is independent of a meiotic program, especially as the meiotic reduction steps were for the most part absent in the stress-induced hybrids. Nevertheless, a similar finding was recently reported in an scRNA-seq analysis of T. brucei stages in the tsetse fly salivary glands, in which the gamete cell cluster was defined based solely on the upregulation of HOP1 and HAP2 ( Hutchinson et al, 2021 ). It is possible that some of these markers of gametogenesis are expressed within a narrow time frame, and by analyzing single-cell gene expression at a single time point post-irradiation, we may have missed their expression peak in what is likely a rare cell population.…”

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

“…Several BARP genes were the prominent marker transcripts in cluster C4 ( Fig 1D ) , identifying this cluster as salivary gland epimastigotes, consistent with previous studies [ 19 , 20 ]. The majority of the later time point (day 40 pi) cells were found in clusters C5 and C6 ( Fig 1B and 1C ), showing that these clusters represent the later salivary gland developmental stages including mature metacyclics and/or their immediate precursors, pre- and nascent metacyclics.…”

“…FHc was also the most significant marker gene for the midgut forms when integrated with T . brucei single-cell data from [ 20 ], supporting the cell-type assignment across datasets ( S4 Fig and S3 Table ) . C1 also expressed several genes encoding surface proteins at high levels ( EP1 : Tb927.10.10260, EP3-2 : Tb927.6.520, and EP2 : Tb927.10.10250), as well as three Proteins Associated with Differentiation ( PAD1 : Tb927.7.5930, PAD2 : Tb927.7.5940, PAD7 : Tb927.7.5990) ( S2 Table ) , which are implicated as sensors of environmental stimuli and trigger differentiation [ 35 ].…”

“…Recent studies have profiled T . brucei populations using single-cell droplet-based approaches from BSF culture to profile the development of stumpy forms [ 18 ] as well as in vivo salivary gland parasites in order to identify a potential vaccine candidate among mature metacyclics [ 19 ] and the dynamics of VSG expression in the developing metacyclic parasites [ 20 ]. These studies complement the previous bulk transcriptomic studies in T .…”

HAP2-Mediated Gamete Fusion: Lessons From the World of Unicellular Eukaryotes

“…While it is unclear whether fertilization/sexual reproduction is obligatory for infectious transmission of these parasites, Trypanosomes undergo a sexual stage in tsetse fly or triatomine insect vectors as evidenced by genetic exchange (Jenni et al, 1986;Gaunt et al, 2003), cytoplasmic mixing between parasites (that is cell-cell fusion) (Gibson et al, 2008), and the expression of conserved meiosis-specific genes prior to cell-cell fusion (Peacock et al, 2011(Peacock et al, , 2014. Recent single-cell RNA-seq studies have demonstrated that T. brucei HAP2 is one of a cluster of gamete-specific genes upregulated in the salivary glands of infected tsetse flies (Hutchinson et al, 2021), and YFP-tagged HAP2 has been shown to be expressed in parasites isolated from tsetse fly salivary glands (Castellanos, 2018). As noted above, the crystal structure of domain II of T. cruzi HAP2 has also been solved and displays overall conservation of structure when compared to other the class II fusion proteins, but also has substantial differences from Chlamydomonas and Arabidopsis HAP2 in the arrangement and structure of its membrane interaction motif (Fedry et al, 2018).…”