Single-cell RNA sequencing of Plasmodium vivax sporozoites reveals stage- and species-specific transcriptomic signatures

Abstract

Background Plasmodium vivax sporozoites reside in the salivary glands of a mosquito before infecting a human host and causing malaria. Previous transcriptome-wide studies in populations of these parasite forms were limited in their ability to elucidate cell-to-cell variation, thereby masking cellular states potentially important in understanding malaria transmission outcomes. Methodology/Principal findings In this study, we performed transcription profiling on 9,947 P. vivax sporozoites to assess the extent to which they differ at single-cell resolution. We show that sporozoites residing in the mosquito’s salivary glands exist in distinct developmental states, as defined by their transcriptomic signatures. Additionally, relative to P. falciparum, P. vivax displays overlapping and unique gene usage patterns, highlighting conserved and species-specific gene programs. Notably, distinguishing P. vivax from P. falciparum were a subset of P. vivax sporozoites expressing genes associated with translational regulation and repression. Finally, our comparison of single-cell transcriptomic data from P. vivax sporozoite and erythrocytic forms reveals gene usage patterns unique to sporozoites. Conclusions/Significance In defining the transcriptomic signatures of individual P. vivax sporozoites, our work provides new insights into the factors driving their developmental trajectory and lays the groundwork for a more comprehensive P. vivax cell atlas.

Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets

“…Furthermore, the median number of UMIs per schizont (6905) was also greater in the current dataset versus other reports. The median number of genes detected per hypnozoite (519) and median number of UMIs per hypnozoite (672) were consistent with other single-cell reports assessing non-replicating, uninucleate stages ( Bogale et al., 2021 ; Real et al., 2021 ; Ruberto et al., 2021 ), including recent single-cell assessments of P. vivax sporozoites ( Ruberto et al., 2022 ) and hypnozoites ( Mancio-Silva et al., 2022 ) ( Figure S3 ). The intact parasitophorous vacuole membrane of hypnozoites in cultures assessed on days 5 and 9 ( Figures 1B , S1 ), the association between infection rates observed by IFA and the number of transcriptomes assessed, and the per cell metrics typical of Plasmodium spp.…”

“…scRNA-seq data sets, revealing highly variable gene expression patterns across, and within, the various stages of the parasite’s life cycle. Here, the number of genes detected, as well as the UMIs, per hypnozoite is in line with other single-cell reports assessing the transcriptomic signatures of non-replicating stages of the parasite’s life cycle ( Howick et al., 2019 ; Bogale et al., 2021 ; Real et al., 2021 ; Ruberto et al., 2022 ), including hypnozoites ( Mancio-Silva et al., 2022 ). Our transcriptome-wide assessment supports the notion that hypnozoites are metabolically active ( Adams and Mueller, 2017 ), demonstrated by the transcription of genes associated with various biological functions including glycolysis and fatty acid metabolism.…”

“…The P. vivax PVP01 transcript information included the nucleotides encoding for the gene-flanking untranslated regions (UTRs) ( Siegel et al., 2020 ), which is important because of the poly-dT capture strategy of the technology used in our workflow. This information partially alleviates the challenges associated with transcription quantification ( Sà et al., 2020 ; Ruberto et al., 2022 ). Post mapping, we used the transcript information (i.e.…”

“… Comparison of per cell metrics with other P. vivax scRNA-seq data. Violin plots showing the distribution of genes detected in the current dataset, and other single-cell gene expression assessments of P. vivax liver stages ( Mancio-Silva et al., 2022 ), sporozoites ( Ruberto et al., 2022 ), and blood-stage parasites ( Sà et al., 2020 ). Replicates in sporozoite and blood-stage datasets represent unique 10x Genomics’ scRNA-seq library preparations.…”

“…In Plasmodium spp., scRNA-seq analyses have so far revealed that individual parasite forms exist on a spectrum of transcriptomic states ( Poran et al., 2017 ; Reid et al., 2018 ; Howick et al., 2019 ; Bogale et al., 2021 ; Real et al., 2021 ; Ruberto et al., 2021 ). Their application to study the biology of P. vivax is a burgeoning area of research as only recently have reports described the transcriptomic signatures of sporozoites and blood stages using 10x Genomics’ technology ( Sà et al., 2020 ; Ruberto et al., 2022 ), and liver forms using Seq-Well S 3 technology ( Mancio-Silva et al., 2022 ).…”