Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum

Abstract

BackgroundMalaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes.ResultsAnalysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates.ConclusionsAnalysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5257-x) contains supplementary material, which is available to authorized users.

Investigating a Plasmodium falciparum erythrocyte invasion phenotype switch at the whole transcriptome level

“…Cultures were initially grown under static culture conditions for the generation of baseline schizont material for analysis, and the sialic acid-dependent invasion phenotype of both strains was confirmed using a previously described procedure 17 . The true positive discovery rate of differentially expressed genes should increase significantly by using a larger number of biological replicates 85 , with six replicates having previously been recommended for differential expression analyses between different strains of Plasmodium falciparum 35 . This study aimed at identifying differentially expressed genes between parasites of the same strain grown under different conditions, thus we projected to study each condition with eight biological replicates.…”

“…Parasites were maintained as 25 ml cultures at 4% hematocrit, and at about 10% parasitemia with more than 50% rings, the ring stages were selected by treatment with 5% sorbitol 86 . These parasites were cultured to develop to schizont stages, the schizonts were purified by percoll-alanine discontinuous gradient centrifugation 17 , diluted with five-fold more fresh erythrocytes, 10 µM E64 added to prevent egress and cultured for a 6-hour period to allow schizont maturation 35 . Mature schizonts were purified by percoll-alanine, homogenized with 500 µL of TRIzol reagent (Ambion/Life Technologies, Carlsbad, California) and stored at −80 °C until processing for RNA extraction.…”

“…This observation also allowed us to confidently use the highly replicated BL cultures as an alternative to ST cultures as comparator against SP cultures in the differential expression analysis. A more stringent adjusted P value (<0.01) 35 was however applied for the transcriptome screen to identify significantly differentially expressed genes in the SP cultures relative to BL cultures.…”

“…Whole mRNA transcriptome library preparation and sequencing was performed using methods as previously described 35 . Briefly, RNA sequencing libraries were prepared with TruSeq Stranded mRNA Library Prep Kit (Illumina) using 500 ng -1 µg RNA.…”

“…We considered the possibility that the phenotypic switch to sialic acid-independent invasion may also involve genes that are not yet known to be associated with different invasion pathways. To this end, we conducted whole transcriptome analysis of schizonts from parasite cultures that had switched invasion pathways during suspension culture, compared with schizonts from unswitched static cultures, using highly biologically replicated samples 35 . This has revealed the differential expression of a larger repertoire of genes likely to be involved in invasion phenotype switching, including known invasion ligands as well as gene families not previously implicated in erythrocyte invasion.…”