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

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.

Sex in protists: A new perspective on the reproduction mechanisms of trypanosomatids

“…These effects revealed the role of DMC1 in the sexual reproduction of this parasite ( Mlambo et al, 2012 ). In T. brucei , the lack of DMC1 activity in repair by homologous recombination and antigenic variation ( Proudfoot and McCulloch 2006 ), combined with high DMC1 expression in sexual stages of the parasite, such as gametes and intermediates ( Peacock et al, 2011 , 2014 , 2021 ; Howick et al, 2021 ), demonstrate the fundamental role of this recombinase in reproduction. These findings underscore the relevance of DMC1 or homologous proteins in meiotic recombination events and suggest that individuals who reproduce sexually contain functional recombinase DMC1 in their genomes.…”

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research

“…scRNA-seq has been used to dissect mixed T. b. brucei populations and identify novel marker genes. These include slender and stumpy bloodstream forms generated in vitro ( Briggs et al., 2021 ) and epimastigotes, gametes and metacyclics found in the tsetse fly salivary glands ( Vigneron et al., 2020 ; Hutchinson et al., 2021 ; Howick et al., 2022 ). Additionally, midgut derived procyclic and proventricular forms have recently been profiled with scRNA-seq ( Howick et al., 2022 ).…”

“…These include slender and stumpy bloodstream forms generated in vitro ( Briggs et al., 2021 ) and epimastigotes, gametes and metacyclics found in the tsetse fly salivary glands ( Vigneron et al., 2020 ; Hutchinson et al., 2021 ; Howick et al., 2022 ). Additionally, midgut derived procyclic and proventricular forms have recently been profiled with scRNA-seq ( Howick et al., 2022 ). If parasites transitioning between broad life cycle forms are also captured, trajectory analysis can be used to order individual parasites according to the gradual change in their transcriptome ( Figure 5 ).…”

“…Differential expression analysis is then used to find dynamic transcript changes during differentiation between life cycle forms. This approach uncovered genes peaking in expression during the slender to stumpy transition, including critical regulator ZC3H20 ( Briggs et al., 2021 ), and highlighted upregulation of transcripts associated with translation and the ribosome during development of both stumpy ( Briggs et al., 2021 ) and metacyclic forms ( Howick et al., 2022 ). Interestingly, scRNA-seq profiling of parasites extracted from tsetse salivary glands highlighted that pre-metacyclics express up to 6 mVSG before selecting just one for monoallelic expression in mature metacyclics ( Hutchinson et al., 2021 ; Howick et al., 2022 ).…”

Parasite OMICS, the grand challenges ahead

“…Similarly, host responses to infections at a single cell level have been documented for bacterial and viral infections, but for parasitology, the initial studies have focused on expression profiles of the parasites at a single cell level. For example, a recent paper used scRNAseq to identify tissue-specific cell types by profiling individual T. brucei trypanosomes from midgut, proventriculus, and salivary glands of infected tsetse flies (Howick et al, 2022). scRNAseq profiles have also been reported for infective and adult stage of S. mansoni (Diaz Soria et al, 2020;Wendt et al, 2020), two important stages of the parasite.…”