Successful Profiling of Plasmodium falciparum
            <i>var</i>
            Gene Expression in Clinical Samples via a Custom Capture Array

Stucke, Emily M.; Dara, Antoine; Dwivedi, Ankit; Hodges, Theresa K; Ott, Sandra; Coulibaly, Drissa; Koné, Abdoulaye K.; Traoré, Karim; Guindo, Boubacary; Tangara, Bourama; Niangaly, Amadou; Daou, Modibo; Diarra, Issa; Tolo, Youssouf; Sissoko, Mody; Tallon, Luke J.; Sadzewicz, Lisa; Zhou, Albert E.; Laurens, Matthew B.; Ouattara, Amed; Kouriba, Bourèma; Doumbo, Ogobara K.; Takala-Harrison, Shannon; Serre, David; Plowe, Christopher V.; Théra, Mahamadou A.; Travassos, Mark A.; Silva, Joana C.

doi:10.1128/msystems.00226-21

“…parasite nucleic acids, enabling multiplex sequencing, may include parasite-specific enrichment steps [40][41][42]. In our recent study, baits targeting the entire parasite genome were employed to capture parasite transcripts from a single-cell barcoded library prior to sequencing.…”

Section: Trends In Parasitologymentioning

confidence: 99%

Single-cell views of the Plasmodium life cycle

Real

¹

,

Mâncio-Silva

²

2022

Trends in Parasitology

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“…RNA-sequencing has the potential to overcome these limitations and provide a better link between var expression and PfEMP1 phenotype in in vitro assays, co-expression with other genes or gene families and epigenetics. While approaches for var assembly and quantification based on RNA-sequencing have recently been proposed ( Wichers et al, 2021 ; Stucke et al, 2021 ; Andrade et al, 2020 ; Tonkin-Hill et al, 2018 ; Duffy et al, 2016 ), these still produce inadequate assembly of the biologically important N-terminal domain region, have a relatively high number of misassemblies, and do not provide an adequate solution for handling the conserved var variants ( Table 1 ).…”

Section: Introductionmentioning

confidence: 99%

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

Andradi-Brown

¹

,

Wichers

²

,

Thien

³

et al. 2024

eLife

1

0

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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.

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“…RNA-sequencing has the potential to overcome these limitations and provide a better link between var expression and PfEMP1 phenotype in in vitro assays, co-expression with other genes or gene families and epigenetics. While approaches for var assembly and quantification based on RNA-sequencing have recently been proposed (Wichers et al, 2021;Stucke et al, 2021;Andrade et al, 2020;Tonkin-Hill et al, 2018;Duffy et al, 2016 ), these still produce inadequate assembly of the biologically important N-terminal domain region, have a relatively high number of misassemblies, and do not provide an adequate solution for handling the conserved var variants (Table 1).…”

Section: Introductionmentioning

confidence: 99%

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

Andradi-Brown

¹

,

Wichers

²

,

Thien

³

et al. 2023

Preprint

View full text Add to dashboard Cite

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.

show abstract

Successful Profiling of Plasmodium falciparum var Gene Expression in Clinical Samples via a Custom Capture Array

Abstract: Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system.

Cited by 5 publications

References 30 publications

Single-cell views of the Plasmodium life cycle

Single-cell views of the Plasmodium life cycle

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

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

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