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

Anthony A Ruberto,Nicolas Merienne,Ivo Mueller,Caitlin Bourke,Caitlin Bourke,Ivo Mueller,Rogerio Amino,Thomas Obadia,Ivo Mueller

doi:10.1038/s41598-021-82914-w

Abstract

In the malaria-causing parasite’s life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite’s development in the mosquito.

Highlights

In the malaria-causing parasite’s life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host
To minimize the risk of sequencing mosquito content, GFP P. berghei ANKA sporozoites dissected from the MIDs and salivary gland (SG) of An. stephensi mosquitoes were purified using a density gradient[30] (Supplementary Fig. S1a online)
In order to assess the reproducibility of the technology and our workflow, we sequenced three mixed MID:SG sporozoite libraries derived from two different mosquito feeds (Fig. 1a; Supplementary Fig. S1b online)

Summary

Introduction

In the malaria-causing parasite’s life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. We use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite’s development in the mosquito. An attractive target for intervention measures is the parasite in the sporozoite stage of its life cycle Before it can infect a mammalian host, a Plasmodium sporozoite must travel from the midgut (MID) of a mosquito to the salivary gland (SG). Despite its high gene coverage, the platebased nature of the protocol makes detection of rare cell populations difficult due to the considerably fewer cells

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Conclusion