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

Sebastian Hutchinson,Roberta Menafra,Aline Crouzols,Andrew D Griffiths,Brice Rotureau,Sophie Foulon,Philippe Bastin

doi:10.1371/journal.ppat.1009904

Sebastian Hutchinson, Roberta Menafra + Show 5 more

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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.

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African trypanosomes are single-cell flagellated parasites that cause Human African Trypanosomiases, and nagana in cattle in sub-Saharan Africa
To test whether trypanosomes would not be visibly stressed by the inDrop pipeline, we monitored the survival and RNA metabolism using ALBA3 and DHH1 as markers for cellular stress in procyclic form (PCF) cells, in conditions which mimic the inDrop protocol
Dimensionality reduction using the Uniform Manifold Approximation and Projection (UMAP) algorithm [45] revealed three clusters which corresponded to the three expected cell types in approximately the expected proportions (Fig 1B), with 164 (8%) Ramos cells, 809 (41%) procyclic cells and 1,006 (51%) bloodstream form (BSF) cells

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Introduction

African trypanosomes are single-cell flagellated parasites that cause Human African Trypanosomiases, and nagana in cattle in sub-Saharan Africa. The parasites have a digenetic life cycle, cycling through a mammalian reservoir host and a tsetse fly vector host. During the developmental phase in the fly, the parasite progresses through multiple stages before acquiring mammalian infectivity in a process called metacyclogenesis in the salivary glands [1]. Trypanosome cells can be classified into multiple stages grouped in two morphotypes: trypomastigote and epimastigote forms (S1 Fig), defined by the relative positions of the nuclear and mitochondrial (kinetoplast) DNA within the cell [2]. Only a small subset of cells eventually differentiates into epimastigote forms during a narrow time frame [3]

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