Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts

Abstract

BackgroundCytoadherence of Plasmodium falciparum-infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50–60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript.MethodsP. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBLα specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.ResultsEach cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR.ConclusionTransfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.

Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor

“…6D). We excluded the possibility that these results were due to expression of var genes coding for ligands of ICAM1 (Golnitz et al ., 2008) by performing real‐time PCR analysis of these particular genes in 3D7_VIR14‐3HA (Fig. S6).…”

Analysis ofPlasmodium falciparum varGenes Expressed in Children from Papua New Guinea

“…We previously reported a statistically signifi ant quantitative shift from var group B to group C transcripts in the same symptomatic and asymptomatic malaria cases, demonstrated by real-time PCR [23]. However, quantitative analysis based on cloning and sequencing of PCR products introduces bias through primers, amplificatio plateaus, and cloning and cannot be compared directly with quantitative PCR [33]. A combination of quantitative and qualitative information about var transcripts provides the most meaningful data.…”

Plasmodium falciparum Variability and Immune Evasion Proceed from Antigenicity of Consensus Sequences from DBL6ε; Generalization to All DBL from VAR2CSA

“…In the 3D7 genome, we did not find any consensus sequences identical to those of its var2csa , but we found a partial sequence of var2csa identical to another PfEMP1 of the 2155-3 genome (accession number: CQ848092). The var gene PFF1580c (accession number: CAG25137.7) also referred to as MAL6P1.6 (Figure S7) is expressed by parasites involved in infections targeting children [44] and is associated with severe disease [45]. Its membrane-proximal domain, DBL7ε, has VB3, VB4 and VB5 homologous to consensus sequences VB3-1, VB4-1 and VB5-4.…”