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


METHODS
P. 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 DBLalpha specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs.


RESULTS
Each 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.


CONCLUSIONS
Transfected 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.
Gölnitz
Uta
U
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Avenida Prof, Lineu Prestes 1374, São Paulo - SP, Brazil. uta.goelnitz@gmx.de
Albrecht
Letusa
L
Wunderlich
Gerhard
G
D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004912:Erythrocytes / Q000469:parasitology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006801:Humans; D016778:Malaria, Falciparum / Q000453:epidemiology; D010641:Phenotype; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000235:genetics* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA
D015800:Protozoan Proteins; C085731:erythrocyte membrane protein 1, Plasmodium falciparum