Single-cell RNA sequencing of Trypanosoma brucei from tsetse salivary glands unveils metacyclogenesis and identifies potential transmission blocking antigens

Abstract

Tsetse-transmitted African trypanosomes must develop into mammalian-infectious metacyclic cells in the fly’s salivary glands (SGs) before transmission to a new host. The molecular mechanisms that underlie this developmental process, known as metacyclogenesis, are poorly understood. Blocking the few metacyclic parasites deposited in saliva from further development in the mammal could prevent disease. To obtain an in-depth perspective of metacyclogenesis, we performed single-cell RNA sequencing (scRNA-seq) from a pool of 2,045 parasites collected from infected tsetse SGs. Our data revealed three major cell clusters that represent the epimastigote, and pre- and mature metacyclic trypanosome developmental stages. Individual cell level data also confirm that the metacyclic pool is diverse, and that each parasite expresses only one of the unique metacyclic variant surface glycoprotein (mVSG) coat protein transcripts identified. Further clustering of cells revealed a dynamic transcriptomic and metabolic landscape reflective of a developmental program leading to infectious metacyclic forms preadapted to survive in the mammalian host environment. We describe the expression profile of proteins that regulate gene expression and that potentially play a role in metacyclogenesis. We also report on a family of nonvariant surface proteins (Fam10) and demonstrate surface localization of one member (named SGM1.7) on mature metacyclic parasites. Vaccination of mice with recombinant SGM1.7 reduced parasitemia early in the infection. Future studies are warranted to investigate Fam10 family proteins as potential trypanosome transmission blocking vaccine antigens. Our experimental approach is translationally relevant for developing strategies to prevent other insect saliva-transmitted parasites from infecting and causing disease in mammalian hosts.

The establishment of variant surface glycoprotein monoallelic expression revealed by single-cell RNA-seq ofTrypanosoma bruceiin the tsetse fly salivary glands

“…We sought to further validate our findings by turning to previously published scRNAseq data of salivary gland trypanosomes using both a different trypanosome strain; RUMP503, and a different technique, the 10xGenomics single-cell RNA-seq system (Vigneron, O'Neill et al 2020). As the complete set of mVSG genes was not available for this strain, we used Trinity (Grabherr, Haas et al 2011) to assemble mVSG transcripts de novo from bulk RNA-seq data (Savage, Kolev et al 2016), and retained the 8 most abundant mVSG genes for downstream analysis.…”

Identification of positive and negative regulators in the stepwise developmental progression towards infectivity in Trypanosoma brucei

“…Thus, the generation of metacyclics relies on a combination of factors that are required for two distinct processes in bloodstream form trypanosome biology: (i) transition to quiescence and (ii) differentiation from bloodstream to procyclic forms. The likely explanation for this observation is that in addition to establishment of quiescence 23 , metacyclic cells also thoroughly remodel their surface with the synthesis of a VSG coat and their transcriptome largely resembles the bloodstream form transcriptome 23,41 . The dominant group of genes that exhibited effects on metacyclogenesis in our RNAi screen were signal transducers ( Fig.…”

Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei

“…For example, at day 2 post-RBP6 OE , 1,221 transcripts were up-regulated and 1,588 transcripts were down-regulated when compared to the uninduced cells (Fig 2A), indicating a global change in transcript expression. First, we analyzed the expression profile of well-known surface molecule markers for early and late PCF (GPEET and EP procyclins, respectively) [29], mature epimastigotes (BARPs) [27], and infectious metacyclics, which express metacyclic-like variable surface glycoproteins (mVSGs) [30]. The GPEET transcript was highly down-regulated, while the procyclin EP transcripts and BARP transcripts were upregulated immediately at day 2 following RBP6 induction.…”