Smedegaard Hansson, H. | Search bioRxiv

bioRxiv2024

Plasmodium falciparum expresses fewer var genes at lower levels during asymptomatic dry season infections than clinical malaria cases

In seasonal transmission areas, clinical malaria occurs during the wet season when mosquitoes are present, while in the dry season, malaria transmission is interrupted and clinical cases are rare. In Mali, Plasmodium falciparum can persist in low parasitaemic asymptomatic individuals through the six-month dry season and shows reduced cytoadhesion of infected erythrocytes, evidenced by the circulation of further developed parasite stages compared to clinical malaria cases. How prolonged circulation of infected erythrocytes is achieved remains unknown. Here, we explored var gene expression in subclinical infections and clinical malaria cases of Malian children, collected during the dry and wet seasons. We sequenced expressed var DBL-tags, used bioinformatic tools to predict their domain composition, binding phenotype and upstream sequence type; and determined their relationship to seasonality and clinical presentation. We found that parasites of asymptomatic infections expressed fewer var genes, with a larger proportion of var transcripts attributed to one or a few vars. In contrast, clinical cases exhibited expression of many var genes at lower proportions. We found that parasites of asymptomatic carriers expressed a mixture of CD36- and EPCR-binding PfEMP1, which changed over time. We confirmed that vars encoding CD36-binding PfEMP1 dominated in non-severe malaria cases, and found no significant difference in expressed var types between dry and wet seasons. Asymptomatic carriers were older, had higher titers of anti-P. falciparum antibodies, and broader reactivity to PfEMP1, suggesting that host immunity was the main determinant limiting var transcript variation in asymptomatic carriers. However, by RNAseq and qRT-PCR we also observed significantly higher total var transcript levels in malaria cases compared to asymptomatic carriers, suggesting that in addition to the parasites switching and the hosts immune selection of expressed var genes, parasites able to sustain long-term infections may be poised for reduced PfEMP1 expression.

microbiology↗

bioRxiv2023

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

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 outperforms the earlier approach of Wichers et al., 2021 on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can 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 of parasite isolates 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. Modest but 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.

bioinformatics↗