ABSTRACT
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. CONCLUSION: 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.
Subject(s)
Gene Expression Profiling , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Animals , CHO Cells , Cell Adhesion , Cloning, Molecular , Cricetinae , Cricetulus , Erythrocytes/parasitology , Gene Expression Regulation , Humans , Malaria, Falciparum/epidemiology , Phenotype , Plasmodium falciparum/isolation & purification , Protozoan Proteins/physiology , Reverse Transcriptase Polymerase Chain Reaction/methods , Sequence Analysis, DNAABSTRACT
The var genes of Plasmodium falciparum code for the antigenically variant erythrocyte membrane proteins 1 (PfEMP1), a major factor for cytoadherence and immune escape of the parasite. Herein, we analyzed the var gene transcript turnover in two ongoing, non-symptomatic infections at sequential time points during two weeks. The number of different circulating genomes was estimated by microsatellite analyses. In both infections, we observed a rapid turnover of plasmodial genotypes and var transcripts. The rapidly changing repertoire of var transcripts could have been caused either by swift elimination of circulating var-transcribing parasites stemming from different or identical genetic backgrounds, or by accelerated switching of var gene transcription itself.
Subject(s)
Antigenic Variation/genetics , Antigens, Protozoan/genetics , Malaria, Falciparum/parasitology , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Adult , Animals , DNA, Protozoan/genetics , Female , Genome, Protozoan , Genotype , Humans , Male , Polymerase Chain Reaction , RNA, Protozoan/genetics , Time Factors , Transcription, Genetic/geneticsABSTRACT
Os genes var de Plasmodium falciparum codificam as proteínas variantes da superfície do eritrócito infectado (PfEMP1). Neste estudo examinamos a mudança de transcritos destes genes var em duas infecções assintomáticas durante um curto prazo e estimamos simultaneamente o número de genomas circulantes nas mesmas amostras por análise de microssatélites. Nas duas infecções observamos uma rápida mudança de genótipos e transcritos de genes var. A mudança acelerada do repertório de transcritos possivelmente foi causada pela rápida eliminação de parasitas circulantes transcrevendo genes var a partir de genomas iguais ou diferentes, ou pela mudança acelerada da própria transcrição (switching) de genes var.