Single-cell transcription analysis of Plasmodium vivax blood-stage parasites identifies stage- and species-specific profiles of expression

Sá, Juliana M.; Cannon, Matthew; Caleon, Ramoncito L.; Wellems, Thomas E.; Serre, David

doi:10.1371/journal.pbio.3000711

Cited by 57 publications

(88 citation statements)

References 76 publications

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“…Single-Cell Transcriptome Analysis. A custom analysis pipeline, similar to the Cell Ranger single-cell software (37), was developed to process all scRNA-seq reads (32). Briefly, we identified all reads containing the 10× Genomics barcodes, trimmed of sequences downstream of 3′ polyadenylation tails, and only kept for further analyses reads longer than 40 bp.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development

Bogale

Pascini

Kanatani

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Despite the critical role of Plasmodium sporozoites in malaria transmission, we still know little about the mechanisms underlying their development in mosquitoes. Here, we use single-cell RNA sequencing to characterize the gene expression profiles of 16,038 Plasmodium berghei sporozoites isolated throughout their development from midgut oocysts to salivary glands, and from forced salivation experiments. Our results reveal a succession of tightly regulated changes in gene expression occurring during the maturation of sporozoites and highlight candidate genes that could play important roles in oocyst egress, sporozoite motility, and the mechanisms underlying the invasion of mosquito salivary glands and mammalian hepatocytes. In addition, the single-cell data reveal extensive transcriptional heterogeneity among parasites isolated from the same anatomical site, suggesting that Plasmodium development in mosquitoes is asynchronous and regulated by intrinsic as well as environmental factors. Finally, our analyses show a decrease in transcriptional activity preceding the translational repression observed in mature sporozoites and associated with their quiescent state in salivary glands, followed by a rapid reactivation of the transcriptional machinery immediately upon salivation.

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Section: Methodsmentioning

confidence: 99%

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development

Bogale

Pascini

Kanatani

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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“…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.…”

Section: Direct-from-host Methodologies Exhibit Distinct Challenges Amentioning

confidence: 99%

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

Brown

Guler

2020

Trends in Parasitology

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Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes. Highlights Standard in vitro culture environments differ dramatically from in vivo conditions in nutrient levels, hematocrit, and rheology and have lower variability in gas levels and temperature. Nutritional and physical differences lead to pronounced, and often rapid, changes in phenomenon, important for understanding virulence in Plasmodium. Parasite drug sensitivity may be altered due to culture adaptation selection, supraphysiological metabolite concentrations, and in vitro media formulations. Parasites propagated in vitro, versus in vivo, show altered transcriptomic and genomic patterns related to virulence factors, metabolism, gametocytogenesis, and more. Direct-from-host methodologies avoid the impacts of in vitro culture adaptation but limit the types of assessments that can be performed as many experiments either require equipment not readily available in endemic settings or necessitate long-term manipulation.

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“…However, these approaches are extremely expensive, and therefore impractical for large scale transcriptomics. More importantly, current 10x transcriptomics protocols produce extremely sparse transcriptomics data from each cell [7, 33]. While these approaches are useful for categorizing parasite to particular lifecycle stages, they provide insufficient resolution for most quantitative genomics applications.…”

Section: Discussionmentioning

confidence: 99%

Efficient transcriptome profiling across the malaria parasite erythrocytic cycle by flow sorting

Dia

Jett

McDew-White

et al. 2020

Preprint

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Plasmodium falciparum is the most virulent and widespread of the human malaria parasite species. This parasite has a complex life cycle that involves sexual replication in a mosquito vector and asexual replication in a human host. During the 48-hour intraerythrocytic developmental cycle (IDC), parasites develop and multiply through the morphologically distinct ring, trophozoite and schizont stages. Stage-specific transcriptomic approaches have shown gene expression profiles continually change throughout the IDC. Cultures of tightly synchronized parasites are required to capture the transcriptome specific to a developmental stage. However, the most commonly used synchronization methods require lysis of late stages, potentially perturbing transcription, and often do not result in tightly synchronized cultures. To produce complete transcriptome profiles of the IDC a synchronous culture requires frequent sampling over a 48-hour period, this is both time consuming and labor intensive. Here we develop a method to sample the IDC densely by isolating parasites from an asynchronous culture with fluorescence activated cell sorting (FACS). We sort parasites in tight windows of IDC progression based on their DNA/RNA abundance. We confirmed the tight synchronization and stage specificity by light microscopy and RNAseq profiling. We optimized our protocol for low numbers of sorted cells allowing us to rapidly capture transcriptome profiles across the entire IDC from a single culture flask. This methodology will allow malaria stage-specific studies to perform experiments directly from asynchronous cultures with high accuracy and without the need for labor-intensive time-course experiments.

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Single-cell transcription analysis of Plasmodium vivax blood-stage parasites identifies stage- and species-specific profiles of expression

Cited by 57 publications

References 76 publications

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development

Transcriptional heterogeneity and tightly regulated changes in gene expression during Plasmodium berghei sporozoite development

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

Efficient transcriptome profiling across the malaria parasite erythrocytic cycle by flow sorting

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