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

Virginia M Howick,Lori Peacock,Wendy Gibson,Mara K N Lawniczak,Clare Collett,Chris Kay

doi:10.1371/journal.ppat.1010346

Abstract

Early diverging lineages such as trypanosomes can provide clues to the evolution of sexual reproduction in eukaryotes. In Trypanosoma brucei, the pathogen that causes Human African Trypanosomiasis, sexual reproduction occurs in the salivary glands of the insect host, but analysis of the molecular signatures that define these sexual forms is complicated because they mingle with more numerous, mitotically-dividing developmental stages. We used single-cell RNA-sequencing (scRNAseq) to profile 388 individual trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies allowing us to identify tissue-specific cell types. Further investigation of salivary gland parasite transcriptomes revealed fine-scale changes in gene expression over a developmental progression from putative sexual forms through metacyclics expressing variant surface glycoprotein genes. The cluster of cells potentially containing sexual forms was characterized by high level transcription of the gamete fusion protein HAP2, together with an array of surface proteins and several genes of unknown function. We linked these expression patterns to distinct morphological forms using immunofluorescence assays and reporter gene expression to demonstrate that the kinetoplastid-conserved gene Tb927.10.12080 is exclusively expressed at high levels by meiotic intermediates and gametes. Further experiments are required to establish whether this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

Highlights

The African tsetse-transmitted trypanosomes are single-celled parasites that cause human and animal diseases, which are a heavy burden for many countries in sub-Saharan Africa
When blood infected with Trypanosoma brucei is imbibed by the tsetse fly, trypanosome blood stream forms (BSF) rapidly change their transcriptional profile, including switching off Variant Surface Glycoprotein (VSG) transcription and upregulating expression of other surface proteins such as procyclins [2,3] They switch their metabolism from dependence on glucose processed via glycolysis in the glycosome to exploitation of amino acids such as proline via the mitochondrial TCA cycle [4]
We found a mean of 2.6x106 mapped reads per cell and a mean detection of 1756 genes per cell (S1A and S1B Fig), which is a greater number of genes per cell than recently published data from Hutchinson et al 2021 [20] that used a droplet-based method on the same parasite stage (1258 genes per cell)

Summary

Introduction

The African tsetse-transmitted trypanosomes are single-celled parasites that cause human and animal diseases, which are a heavy burden for many countries in sub-Saharan Africa. These trypanosomes survive in both the tsetse and mammalian host by taking on distinct morphological forms that suit the diverse metabolic and immune environments they encounter [1]. When blood infected with Trypanosoma brucei is imbibed by the tsetse fly (genus Glossina), trypanosome blood stream forms (BSF) rapidly change their transcriptional profile, including switching off Variant Surface Glycoprotein (VSG) transcription and upregulating expression of other surface proteins such as procyclins [2,3] They switch their metabolism from dependence on glucose processed via glycolysis in the glycosome to exploitation of amino acids such as proline via the mitochondrial TCA cycle [4]. The sexual cycle appears to be a sideshow in the normal mitotic developmental program, as it occurs in clonal trypanosome lines and does not need to be triggered by external factors such as the presence of another strain

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