A novel computational pipeline for var gene expression augments the discovery of changes in the Plasmodium falciparum transcriptome during transition from in vivo to short-term in vitro culture

Abstract

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that even outperforms the earlier approach of Wichers et al., 2021 on both laboratory and clinical isolates across a combination of metrics. It is a powerful tool to interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

Novel single-cell preservation and RNA sequencing technology unlocks field studies forPlasmodiumnatural infections

“…While laboratory strains are essential for studying Plasmodium biology, they are not necessarily representative of circulating parasite populations. Genome-wide transcriptomic studies have demonstrated inherent differences in gene expression between culture-adapted strains and natural infections (23)(24)(25)(26). Moreover, natural infections are often complex with individuals frequently carrying multiple clones (27)(28)(29)(30)(31).…”