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Single-cell RNA sequencing of Plasmodium vivax sporozoites reveals stage- and species-specific transcriptomic signatures [1]

['Anthony A. Ruberto', 'Department Of Parasites', 'Insect Vectors', 'Institut Pasteur', 'Paris', 'Caitlin Bourke', 'Division Of Population Health', 'Immunity', 'Walter', 'Eliza Hall Institute Of Medical Research']

Date: 2022-08 

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.

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.

Plasmodium vivax is the second most common cause of malaria worldwide. It is particularly challenging for malaria elimination as it forms both active blood-stage infections, as well as asymptomatic liver-stage infections that can persist for extended periods of time. The activation of persister forms in the liver (hypnozoites) are responsible for relapsing infections occurring weeks or months following primary infection via a mosquito bite. How P. vivax persists in the liver remains a major gap in understanding of this organism. It has been hypothesized that there is pre-programming of the infectious sporozoite while it is in the salivary-glands that determines if the cell’s fate once in the liver is to progress towards immediate liver stage development or persist for long-periods as a hypnozoite. The aim of this study was to see if such differences were distinguishable at the transcript level in salivary-gland sporozoites. While we found significant variation amongst sporozoites, we did not find clear evidence that they are transcriptionally pre-programmed as has been suggested. Nevertheless, we highlight several intriguing patterns that appear to be P. vivax specific relative to non-relapsing species that cause malaria prompting further investigation.

Funding: This work was supported by the Agence Nationale de la Recherche ( https://anr.fr , ANR-17-CE13-0025 to A.A.R, G.S., I.M.), the National Health and Medical Research Council of Australia ( www.nhmrc.gov.au , NHMRC; 1092789 and 1134989 to I.M.), National Institute of Allergy and Infectious Diseases ( www.nih.niaid.gov , 5U19AI129392 to I.M), and a NHMRC Principal Research Fellowship ( www.nhmrc.gov.au , 1155075 to I.M). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: All raw sequencing data generated from P. vivax sporozoites in this study are accessible in the European Nucleotide Archive ( www.ebi.ac.uk/ena/ ) under the Bioproject ID: PRJEB42435. P. vivax blood-stage scRNA-seq data were downloaded from NCBI's Short Read Archive (Bioproject ID: PRJNA603327). P. falciparum sporozoite scRNA-seq data were obtained from: https://github.com/vhowick/pf_moz_stage_atlasScripts and supporting files are available on GitHub: https://github.com/BourkeCaitlin/Pv-Spz-singleCell Archived scripts and output files as at time of publication are available on Zenodo: http://doi.org/10.5281/zenodo.6474355

Copyright: © 2022 Ruberto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In this study, we analyse the transcriptomes of 9,947 P. vivax sporozoites captured using droplet-based scRNA-seq technology. We first cross-reference the data with sporozoite bulk microarray and RNA-seq data to show consistent transcription of known genes upregulated in sporozoites. Next, we represent the data in low dimensional space and identify sporozoites in various transcriptomic states using both clustering and pseudotime trajectory methods. Finally, we perform comparative analyses with publicly available P. falciparum sporozoite and P. vivax blood-stage scRNA-seq data [ 23 , 26 ] and highlight both conserved and unique gene usage patterns between sporozoites and erythrocytic forms. Overall, our work provides an important, new resource for the malaria community by offering key insights into gene usage among P. vivax sporozoites and the factors driving their developmental trajectory at a resolution unattainable with bulk transcriptomics.

Single-cell RNA sequencing methods (scRNA-seq) constitute a recent advancement applicable for assessing parasite-to-parasite differences. ScRNA-seq has differentiated multiple transcriptomic states among individual P. berghei and P. falciparum sporozoites [ 22 – 25 ]. However, the extent to which P. vivax sporozoites vary at the single-cell level has not been studied. Therefore, the application of scRNA-seq technology provides an opportunity to explore heterogeneity amongst P. vivax sporozoites and examine the existence of distinct transcriptional signatures that may help better understand the sporozoite’s developmental fate.

The factors underlying the development of P. vivax sporozoites into replicating schizonts or their persistence as hypnozoites and subsequent activation remain poorly defined. Key questions regarding the regulation of hypnozoite biology have focused on how it differs regionally, seasonally, and between strains. Relapse frequency varies by climate and geographical region, with temperate strains exhibiting long periods of latency and tropical ones relapsing at shorter intervals [ 13 – 15 ]. The observation that P. vivax may be able to regulate hypnozoite formation in accordance with environmental conditions feeds into a hypothesis that the developmental outcome within the liver may be pre-determined in the sporozoite [ 16 , 17 ]. In addition, observations in humanised rodent livers have identified sympatric P. vivax strains with stable differences in hypnozoite formation rates [ 18 ]. This points to genetic heterogeneity among P. vivax sporozoites that may play a role in defining developmental fate, consistent with the tachy- and bradysporozoites proposed by Lysenko et al. [ 16 ]. System-wide studies offer an opportunity to find evidence for sporozoite pre-programming; however, previous analyses of P. vivax sporozoites have been performed using bulk-sequencing approaches [ 19 – 21 ] which obscure variation that might exist between individual parasites.

Malaria remains the most significant parasitic disease of humans globally, causing an estimated 229 million infections and 409,000 deaths per year [ 1 ]. Plasmodium spp. are the etiological agents of malaria, and at least five species are known to infect humans [ 1 ]. Plasmodium falciparum and P. vivax are the most prevalent, and both contribute significantly to the malaria disease burden [ 2 – 4 ]. Plasmodium spp. infection in humans begins with the deposition of sporozoites into the dermis when an infected Anopheles mosquito takes a blood meal [ 5 ]. While sporozoites must undergo replication in the liver before mounting a blood-stage infection, P. vivax sporozoites can develop into either a replicating or persisting (hypnozoite) form [ 6 ]. Hypnozoites can remain in the liver for weeks, months or years before activating to undergo schizogony [ 7 ], leading to a relapsing blood-stage infection. Relapsing infections are estimated to comprise up to 90% of P. vivax malaria cases in some regions [ 8 – 10 ]. Relapse-causing hypnozoites, in addition to a high prevalence of sub-detectable and often asymptomatic blood-stage infections, severely limit efforts to eradicate P. vivax malaria [ 3 , 11 ]. Recent modelling suggests that eliminating P. vivax malaria is not possible without programs that specifically target and cure hypnozoite infections [ 12 ].

Results

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[1] Url: https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0010633

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