Transcriptome-module phenotype association study implicates extracellular vesicles biogenesis in Plasmodium falciparum artemisinin resistance

The role of extracellular vesicles in developing artemisinin resistance in Plasmodium falciparum is not known. We have posited a role for EVs in the development of ARTr (EVs export hypothesis) (Tandoh et al.,2022).The objective of this study was to test a prediction of the EVs export hypothesis to gain insight into a putative role for EVs in the expression of the artemisinin resistant phenotype. We compared the gene expression profile of two artemisinin resistant parasites (C580R and R539T) generated by CRISPR based editing of an artemisinin susceptible parasite (Dd2) for enrichment with an EVs module of interest using gene set enrichment analysis. Overall design: We leveraged high throughput next generation sequencing technology to determine the global gene expression profiles of three parasite line (Dd2,R539T, C580R) in unsynchronised cultures. Samples were collected for three conditions: untreated, treated with 0.1% DMSO for six hours and treated with dihydroartemisinin for six hours. At least three independent experiments were conducted for each parasite strain and condition, except for the untreated condition ( which was done in duplicates). The TRIzol method was used to extract total RNA from TRIzol-preserved parasites after thawing at room temperature (Invitrogen). The extracted RNA was dissolved in 100µl RNase-free water and DNA removed by DNase I digestion, after which the purified DNA was eluted into 50µl RNase-free water. Qubit High Sensitivity RNA assay (Thermo Fisher Scientific) was used to quantify the purified RNA, and RNA integrity was determined on an Agilent Bioanalyzer (Agilent Genomics, Waldbronn, Germany). Library preparation and next-generation sequencing for whole mRNA transcriptome were done as per Single Cell Discoveries (The Netherlands) protocols. ----------------------- Single Cell Discovery, Netherlands.