Use of single-cell RNAseq for identification and characterization of sexual stages of Plasmodium knowlesi

Studies of sexual stages of Plasmodium falciparum in vitro have revealed key processes by which gametocytes develop and transmit human infections to Anopheles mosquitoes. However, other species of human malaria, such as Plasmodium vivax and Plasmodium knowlesi, have gametocytes with different biologies, different morphologies, faster development and shorter lifespan compared to P. falciparum, reflecting the evolutionary separation of these species. Asexual stages of P. knowlesi were adapted for in vitro culture in 2002, but gametocytes were not observed in these cultures. P. knowlesi gametocytes were recently generated in vitro and used to infect Anopheles mosquitoes. It was observed that P. knowlesi infections can be transmitted to mosquitoes even in the presence of very low numbers of gametocytes observed in microscopy (often undetectable). Furthermore, transcripts of P. falciparum-specific orthologous markers could not be correlated with P. knowlesi mosquito infectivity markers, indicating that different genes are related to gametocyte development in these species, confirming the difference between gametocyte biology. To identify gametocytes from in vitro cultures and characterize genes and pathways involved in gametocyte development, this project will use single cell RNA sequencing (scRNA-seq). scRNA-seq libraries will be prepared using the ddSEQ Single-Cell-Isolator (BioRad) and the SureCell WTA 3’Library Prep kit (Illumina). The libraries will be sequenced on the Illumina NovaSeq 6000 system (Illumina). The bioinformatics analyzes will be carried out in collaboration with Prof. David Serre (University of Maryland, Baltimore, USA). Dr. Serre is a pioneer in RNA-seq and scRNA-seq analysis of microorganisms, especially Plasmodium. 

Project details

Genetics and Genomics

Nov 2021 — Jan 2022

Brazil