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 characterised 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. We speculate that this protein, currently of unknown function, plays a role in gamete formation and/or fusion.

Single-cell transcriptomics highlights sexual cues among reproductive life stages of uncultivated Acantharia (Radiolaria)

“…Arabidopsis , Fedry et al, 2018), invertebrates (e.g. Drosophila , Hernández and Podbilewicz, 2017) and gamete fusion in many protists such as the green microalgae Chlamydomonas (Archaeplastida, Liu et al, 2008), the ciliate Tetrahymena (Alveolata, Pinello et al, 2023), the social amoeba Dictyostelium (Amoebozoa, Okamoto et al 2016) and the parasites Trypanosoma (Excavata, Howick et al 2021) and Plasmodium (Alveolata, Liu et al 2008). Its wide distribution across eukaryotic lineages with divergent life cycles suggests that HAP2/GCS1 is an ancestral gamete fusogen of eukaryotes (Speijer et al, 2015).…”

“…Concentrating efforts towards culturing Radiolaria would represent a tremendous advance in understanding their life cycle. In addition to facilitating the documentation of additional developmental stages within their sexual life cycle, it would also enable the collection of a sufficient number of single-cell replicates for statistical analysis, as has been done for Plasmodium and Trypanosoma parasites (Howick et al, 2019; Howick et al, 2021). Moreover, establishing a radiolarian culture would significantly accelerate the generation of radiolarian genomes, enabling the validation of recovered transcript sequences and providing a reference framework for functional annotation.…”

“…Meanwhile, lineage specific variations of reference sexual cycle genes have been documented among some protist lineages (Malik et al, 2008; Thangavel et al, 2023). Subsequently, even though the expression of reference sexual cycle genes stands as direct evidence for the existence of a sexual cycle, the presence of sex-related genes requires confirmation through approaches such as in situ hybridisation in putative meiotic stages (Tekle et al, 2020) and gametes (Howick et al, 2021). This would also provide a comprehensive assessment of whether sequence conservation is a relevant proxy for function conservation in the lineage (Maciver et al, 2019).…”

“…For instance, despite cyst life stages being frequently associated with resting stages, there are many cases where cysts have been reported to play a role in protist sexual cycles (e.g., diatoms and dinoflagellates (Archibald et al, 2017; Montagnes et al, 2011). Even among well studied, cultivable pathogenic protists, such as trypanosomes and plasmodiums, novel sexual life stages have been recently identified by functional analysis of genetic data (Howick et al, 2019; Howick et al, 2021).…”