Single-cell transcriptomics to define Plasmodium falciparum stage-transition in the mosquito midgut

Abstract: Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 hours post blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors, and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early-to-late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives.

This repository contains original data such as raw count matrices, metadata files, images parameters, GO term files, etc. isolated time points of P.falciparum single parasite from mosquito midgut experiments using micro-manipulation performed in this study. These folders contain input data for the R markdown script, which can be found in this GitHub repository: Github link. 

The  files contains all data necessary to reproduce the results of the study and have the expression matrix reads counts and metadata to reproduce the data of this study can also be found in the same GitHub repository (https://github.com/ANKARKLEVLAB/Single-cell-P.falciparum-midgut).

for data interactive Visualization please go to ScRNA-seq data Visualization  

Experiment design: Plasmodium falciparum NF54 gametocyte producing cell line were committed to sexual stages (gametocytes) and pooled to 1% gametocytaemia and 50% hematocrit. Gametocyte were membrane fed to Anopheles gambiae adult mosquito and dissected at time points; 2, 4, 8, 12 and 20 hours post infection, parasites were then isolated using Micro-maniupulation based on Anti-Pfs25 antibody conjugated to Alexa-488 flour and  Hoechst stain. cDNA library were prepared using SMART-seq2 and illumina sequenced. ScRNA-seq data were then analyzed using various computational tools, including Seurat toolkit and Slingshot for Pseudotime analyses. 

directories structure:

Processed_datafiles.zip:    

1. metadata.txt (cell IDs + clusters + Time points + Pseudotime modules) 
2. zygote_raw_expressionmatrix.txt (columns = cell Ids , rows = gene accession number) 
3. img_data.txt ( imaging data ,The fluorescence intensity + Volume)
4. metadata_NFFG.txt ( cell IDs + clusters + Time points including unfertilized female gametes) 
5. metadata_without_NFFG.txt (cell IDs + clusters + Pseudotime modules without unfertilized female gametes)