Latent transcriptional variations of individual Plasmodium falciparum uncovered by single-cell RNA-seq and fluorescence imaging

Abstract

Malaria parasites follow a complex life cycle that consists of multiple stages that span from the human host to the mosquito vector. Among the species causing malaria, Plasmodium falciparum is the most lethal, with clinical symptoms manifesting during the intraerythrocytic developmental cycle (IDC). During the IDC, P. falciparum progresses through a synchronous and continuous cascade of transcriptional programming previously established using population analyses. While individual parasites are known to exhibit transcriptional variations to evade the host immune system or commit to a sexual fate, such rare expression heterogeneity is largely undetectable on a population level. Therefore, we combined single-cell RNA-sequencing (scRNA-seq) on a microfluidic platform and fluorescence imaging to delineate the transcriptional variations among individual parasites during late asexual and sexual stages. The comparison between asexual and sexual parasites uncovered a set of previously undefined sex-specific genes. Asexual parasites were segregated into three distinct clusters based on the differential expression of genes encoding SERAs, rhoptry proteins, and EXP2 plus transporters. Multiple pseudotime analyses revealed that these stage-specific transitions are distinct. RNA fluorescent in situ hybridization of cluster-specific genes validated distinct stage-specific expression and transitions during the IDC and defined the highly variable transcriptional pattern of EXP2. Additionally, these analyses indicated huge variations in the stage-specific transcript levels among parasites. Overall, scRNA-seq and RNA-FISH of P. falciparum revealed distinct stage transitions and unexpected degrees of heterogeneity with potential impact on transcriptional regulation during the IDC and adaptive responses to the host.

Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples

“…While the method appears to work relatively well in large, cultured mammalian cells, its limits become in particular apparent when working with smaller cells such as yeast and many protozoans, that have a cell wall or dense cytoskeleton. In trypanosomes for example, there is a huge variance in fluorescence intensities between mRNA molecules within the same cell ( 2 , 3 ) and the same is observed on recent smFISH images from Plasmodium ( 4 ). Almost no yeast publications exist that use branched DNA technology.…”

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

“…For unbiased exploration of parasite samples, 'omics-level investigations offer an increasing breadth of analysis capabilities. Progressively less material has been required to obtain high quality 'omics results with research groups successfully performing RNA-seq [67][68][69] and whole-genome sequencing [70][71][72][73] on single P. falciparum and P. vivax parasites. Single-cell approaches are attractive not only because they require less material, which is conducive to ex vivo/in vivo sampling, but they also have the ability to capture within-population heterogeneity that is frequently missed when analyzing the bulk population.…”

Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites

“…Recent advances in single-cell RNA-seq (scRNA-seq) methods have prompted new ways of deriving biological insights unattainable by bulk RNA-seq efforts 14 , 15 . However, the use of scRNA-seq to explore gene expression patterns across the different Plasmodium parasites is still a nascent area of research 16 – 23 . Only two studies 17 , 20 have previously assessed the transcriptional profiles of MID and SG sporozoites at single-cell resolution.…”