Dataset Information

Var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2.

ABSTRACT:

Background

The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ?60 var genes.

Methods

The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorescence.

Results

Transcripts from var1 (FCR3S1.2(var1); IT4var21) and other var genes were detected by semi-quantitative PCR but results from qPCR showed that one var gene transcript dominated over the others (FCR3S1.2(var2); IT4var60). Antibodies raised in rats to the recombinant NTS-DBL1? of var2 produced in E. coli completely and dose-dependently disrupted rosettes (?95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2(var2) encodes the dominant PfEMP1 expressed in this parasite.

Conclusion

The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2(var2) (IT4var60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1? of FCR3S1.2(var1) is likely due to cross-reactivity with NTS-DBL1? of the var2 encoded PfEMP1.

SUBMITTER: Albrecht L

PROVIDER: S-EPMC3036667 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

var gene transcription and PfEMP1 expression in the rosetting and cytoadhesive Plasmodium falciparum clone FCR3S1.2.

Albrecht Letusa L Moll Kirsten K Blomqvist Karin K Normark Johan J Chen Qijun Q Wahlgren Mats M

Malaria journal 20110125

<h4>Background</h4>The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 var genes.<h4>Methods</h4>The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantit ...[more]

PMID: 21266056

Similar Datasets

Project description:BackgroundRosette-formation of Plasmodium falciparum parasitized erythrocytes is of importance in the development of severe malaria. The parasite-derived molecule PfEMP1 (Plasmodium falciparum erythrocyte membrane protein 1), central to rosetting, is suggested to be included in a multimeric vaccine targeting severe disease.MethodsThree recombinant NTS-DBL1?-domains of PfEMP1 were generated in Escherichia coli, purified and used for immunization of rats and goats. Antibody titres were determined in ELISA assays and responses were compared in-between different individual animals and species. Reactivity with the parasites was tested in live pRBC using FACS. B-cell epitopes prediction was carried out in silico and compared to the results obtained by peptide microarray. Screening for serological cross-reactivity with heterologous NTS-DBL1? variants was carried out by ELISA, peptide array and FACS on pRBC of different laboratory strains and patient isolates.ResultsAll three NTS-DBL1?-domains induced high titres of antibodies that were biologically active with no apparent difference between constructs covering slightly different parts of the DBL1?-sequence. The different animal species showed comparable titres of antibodies, while variations within individuals of the species could be observed.Mapping of the recognized epitopes revealed that most parts of the molecule were able to induce an antibody response with a tendency for the N and C terminal parts of the molecule for slightly higher recognition. Important differences to the epitopes predicted were found as some of the most conserved parts of the DBL1?-domain contained the main epitopes for antibody reactivity. ELISA assays and peptide microarray demonstrated substantial cross-reactivity to heterologous variants, while binding to native PfEMP1 was observed only in few combinations on the pRBC surface, underlining that mainly internal, conserved and not surface exposed parts of the DBL1?-domain are responsible for this observation.ConclusionBiologically active antibodies can be induced consistently, with high titres, in different animal species and the antibodies elicited by different constructs react with similar epitopes. Induced antibodies recognize epitopes localized in all subdomains of the DBL1?-sequence. Cross-reactivity between NTS-DBL1?-variants is common in ELISA, but rare with live pRBC emphasizing that also internal, conserved areas of PfEMP1 carry important highly immunogenic epitopes of the molecule.

Project description:Background: The cytoadherence of Plasmodium falciparum is thought to be mediated by variant surface antigens (VSA), encoded by var, rif, stevor and pfmc-2tm genes. The last three families have rarely been studied in the context of cytoadherence. As most VSA genes are unique, the variability among sequences has impeded the functional study of VSA across different P. falciparum strains. However, many P. falciparum genomes have recently been sequenced, allowing the development of specific microarray probes to each VSA gene. Methods: All VSA sequences from the HB3, Dd2 and IT/FCR3 genomes were extracted using HMMer. Oligonucleotide probes were designed with OligoRankPick and added to the 3D7-based microarray chip. As a proof of concept, IT/R29 parasites were selected for and against rosette formation and the transcriptomes of isogenic rosetting and non-rosetting parasites were compared by microarray. Results: From each parasite strain 50-56 var genes, 125-132 rif genes, 26-33 stevor genes and 3-8 pfmc-2tm genes were identified. The ability of the VSA-supplemented microarray chip to detect cytoadherence-related genes was assessed using P. falciparum clone IT/R29, in which rosetting is known to be mediated by PfEMP1 encoded by ITvar9. Whole transcriptome analysis showed that the most highly upregulated gene in rosetting parasites was ITvar9 (19 to 429-fold upregulated over six time points). Only one rif gene (IT4rifA_042) was upregulated by more than 4-fold (5-fold at 12 hours post-invasion), and no stevor or pfmc-2tm genes were upregulated by more than 2-fold. 49 non-VSA genes were upregulated in rosetting parasites by more than 3-fold in at least two time-points, although none as markedly as ITvar9. Conclusions: We demonstrate that the VSA of newly sequenced P. falciparum strains can be added to the 3D7-based microarray chip, allowing the analysis of the entire transcriptome of multiple strains. For the rosetting clone IT/R29, the striking transcriptional upregulation of ITvar9 was confirmed, and the data did not support the involvement of other VSA families in rosette formation.