Use of single-cell RNAseq technology to identify and characterize Plasmodium knowlesi sexual stages

Abstract

In vitro studies of Plasmodium falciparum sexual stages have revealed key processes by which gametocytes develop and transmit human infections to Anopheles mosquitoes. However, other human malaria species, such as Plasmodium vivax and Plasmodium knowlesi, exhibit distinct gametocyte biology, with different morphology, faster development and shorter lifespan compared to P. falciparum, reflecting the evolutionary separation of these species.Asexual blood stages of P. knowlesi were adapted to in vitro culture in 2002, but gametocytes were not observed in the cultures. Recently, we generate P. knowlesi gametocytes in vitro and have used these parasites to infect Anopheles mosquitoes. We observed that P. knowlesi infections can be transmitted to mosquitoes even in the presence of very low numbers of gametocytes observed by microscopy (often undetectable). Furthermore, transcription of specific P. falciparum gametocyte markers orthologs did not correlate with P. knowlesi infectivity to mosquitoes, indicating that different genes are related to gametocyte development in these species, confirming the differences in gametocyte biology.To identify gametocytes from in vitro cultures and characterize genes and pathways involved in gametocyte development, we will use single-cell RNA sequencing (scRNAseq). Sc-RNAseq libraries will be prepared using the ddSEQ Single-Cell-Isolator (BioRad) and the SureCell WTA 3'Library Prep Kit (Illumina). Libraries will be sequenced in the Illumina NovaSeq 6000 system (Illumina). Bioinformatic analysis will be performed in collaboration with the group of Prof. David Serre (University of Maryland, Baltimore, USA). Dr. Serre is a pioneer in RNAseq and scRNAseq analysis of microorganisms, especially Plasmodium. For this step we are requesting funding for the Master's candidate Taís Baruel Vieira to stay 2 months in Baltimore, to receive bioinformatics training in the UMD at Serre's lab. (AU)