Characterization of Plasmodium vivax transcriptome by Single Cell RNA-sequencing in a non-human primate model

Abstract

Malaria is a significant cause of morbidity and mortality worldwide, responsible for 241 million clinical cases and 627,000 deaths in 2020 according to the World Health Organization. Plasmodium vivax infections, while less deadly than the more widely studied Plasmodium falciparum, is a significant risk to many people in SE Asia and South America. P. vivax infections often consist of heterogenous populations of parasites at different developmental stages and with distinct transcriptional profiles, which complicates gene expression analyses. The advent of single cell RNA sequencing (scRNA-seq) enabled disentangling this complexity and has provided robust and stage-specific characterization of Plasmodium gene expression. However, scRNA-seq information is typically derived from the end of each mRNA molecule (usually the 3’-end) and therefore fails to capture the diversity in transcript isoforms documented in bulk RNA-seq data. P. vivax infections also often contain multiple, genetically-distinct, parasites but the consequences of this polyclonality on the regulation of asexual parasites, their sexual differentiation, and the transmission to a new host remain unknown. Here, we describe the sequencing of P. vivax scRNA-seq libraries obtained from a non-human primate model using traditional Illumina sequencing and Pacific Biosciences (PacBio) chemistry to characterize full-length Plasmodium vivax transcripts from single cell parasites, as well as experimental infections with the NIH-1993-F3 and the Chesson strains of P. vivax. Our results show that many P. vivax genes are transcribed into multiple isoforms, primarily through variations in untranslated region (UTR) length or splicing, and that the expression of many isoforms is developmentally regulated. Our experimental coinfection experiments revealed limited polyclonality when the strains were inoculated one after the other, but yielded robust polyclonal infections by simultaneous infection. In contrast to our hypothesis, this polyclonality did not seem to modify the regulation of individual parasites, or their sexual commitment. Overall, this work yielded unique insights on the mechanisms regulating the establishment of polyclonal P. vivax infections, and their consequences for disease transmission, and provided a validated framework to further study of Plasmodium polyclonal infections.