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

Abstract

the central role that erythrocyte invasion plays in Plasmodium falciparum survival and reproduction makes this process an attractive target for therapeutic or vaccine development. However, multiple invasion-related genes with complementary and overlapping functions afford the parasite the plasticity to vary ligands used for invasion, leading to phenotypic variation and immune evasion. Overcoming the challenge posed by redundant ligands requires a deeper understanding of conditions that select for variant phenotypes and the molecular mediators. While host factors including receptor heterogeneity and acquired immune responses may drive parasite phenotypic variation, we have previously shown that host-independent changes in invasion phenotype can be achieved by continuous culturing of the W2mef and Dd2 P. falciparum strains in moving suspension as opposed to static conditions. Here, we have used a highly biologically replicated whole transcriptome sequencing approach to identify the molecular signatures of variation associated with the phenotype switch. The data show increased expression of particular invasion-related genes in switched parasites, as well as a large number of genes encoding proteins that are either exported or form part of the export machinery. The genes with most markedly increased expression included members of the erythrocyte binding antigens (EBA), reticulocyte binding homologues (RH), surface associated interspersed proteins (SURFIN), exported protein family 1 (EPF1) and Plasmodium Helical Interspersed Sub-Telomeric (PHIST) gene families. the data indicate changes in expression of a repertoire of genes not previously associated with erythrocyte invasion phenotypes, suggesting the possibility that moving suspension culture may also select for other traits.Plasmodium falciparum malaria remains a major global public health challenge 1-3 . The 48-hour cyclical asexual replication of the blood stage parasite is responsible for the clinical symptoms of the infection 4 . Parasite control is hampered by genetic and phenotypic variations that impact negatively on drug and vaccine development strategies. Thus, a better understanding of the molecular mechanisms responsible for parasite phenotypic variation is important for the development and application of new malaria control strategies.Erythrocyte invasion by P. falciparum merozoites has been a subject of significant research interest due to its central role in parasite survival and transmission 5-7 . Some of these studies have demonstrated the importance of invasion-related gene families in the parasite genome, particularly the erythrocyte binding antigens (EBAs) and reticulocyte binding-like homologues (RHs) 5,8-16 . The array of different genes involved in invasion allows the parasite to vary ligands used for invasion [17][18][19][20] , enabling adaptation to differences in host environments including erythrocyte receptor heterogeneity and ligand-specific immune responses [21][22][23][24][25] . Expression and usage of particular ligands appear to depe...

Actomyosin forces and the energetics of red blood cell invasion by the malaria parasite Plasmodium falciparum

“…PfMyoA-K764E was confirmed to have a moderate phenotype after RAP treatment under static conditions while suspension conditions lessened the defect, consistent with the hypothesis that the interaction between phospho-S19 and K764 is critical only in stages where gliding is required [25]. The culture conditions that best imitate physiological conditions are not clear, but suspension culture significantly affects invasion phenotypes and adhesin expression [15,53,54]. The recent demonstration that merozoites exhibit actin-dependent gliding [3] might explain why PfMyoA-K764E had a stronger effect on static invasion, if merozoites first tune PfMyoA for gliding before S19 is dephosphorylated to tune the motor for invasion ( Fig 7C).…”

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

“…A transcriptomics study by Nyarko et al, utilizing parasites grown statically or in suspension, revealed that over 200 genes were differentially expressed between conditions, largely representing increases in erythrocyte-binding, rigidity, and invasion-related genes in suspended cultures [32]. These properties enhance P. falciparum cytoadherence; thus, studying these phenomena in a static environment plausibly leads to an underappreciation of genes relevant to successful cytoadherence and immune evasion under the shear stress of the vascular system in vivo.…”

“…The density of PfEMP1 knobs on the RBC surface ( Figure 1B), which mediate sequestration in P. falciparum to avoid physical filtration by the spleen, was reduced fivefold upon adaptation of ex vivo isolates to in vitro culture [31]. Transcription of many P. vivax cytoadherence genes was reduced in vivo in splenectomized Aotus monkeys relative to spleen-intact monkeys, demonstrating that Plasmodium cytoadherence is likely regulated by many factors, including serum lipoproteins [16], rheology [32], and splenic pressure [33].…”

“…Many host factors are likely responsible for effecting such a dynamic response in parasite antigen transcription, including immunity status and heterogeneity of receptors [55]. Less intuitively, the static nature of most in vitro culture systems has recently been shown to decrease expression of these exported RBC antigens relative to suspension cultures, indicating an important role for fluid flow in vivo [32]. For example, increased expression in a number of ApiAP2 transcription factors has been observed in vivo/ex vivo versus in vitro [47,52,56]; these expression changes appear to be, in part, induced by fluid flow as similar patterns of ApiAP2 upregulation were observed in suspension versus static cultures [32].…”

“…Less intuitively, the static nature of most in vitro culture systems has recently been shown to decrease expression of these exported RBC antigens relative to suspension cultures, indicating an important role for fluid flow in vivo [32]. For example, increased expression in a number of ApiAP2 transcription factors has been observed in vivo/ex vivo versus in vitro [47,52,56]; these expression changes appear to be, in part, induced by fluid flow as similar patterns of ApiAP2 upregulation were observed in suspension versus static cultures [32]. Of note, the gametocyte conversion ApiAP2 transcription factor, ap2-g, as well as other sexual stagerelated genes, is among those seen downregulated in culture conditions [32,47,52,56].…”