Surface Co-Expression of Two Different PfEMP1 Antigens on Single Plasmodium falciparum-Infected Erythrocytes Facilitates Binding to ICAM1 and PECAM1

Louise Joergensen,Gerald K K Cham,Anja T R Jensen,Louise Turner,David E Arnot,Sanne S Berger,Thor G Theander,Michael B Dalgaard,Thomas Lavstsen,Dominique C Bengtsson,Anja Bengtsson,Elena Ronander,Michala E Victor

doi:10.1371/journal.ppat.1001083

Abstract

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required.

Highlights

Plasmodium falciparum is the most pathogenic human malaria parasite and a major cause of morbidity and mortality in Africa
Plasmodium falciparum is the most pathogenic human malaria parasite and its virulence has been linked to its capacity to express different adhesion proteins that enable the developing parasitized erythrocyte to bind to capillaries of the host, thereby avoiding removal by the spleen
Nuclear run-on assays and some single-cell RT-PCR experiments clearly demonstrated transcription of several var genes in ring stages, sometimes [16,17,18], but not always showing a reduction in the extent of polygenic var transcription as rings develop into trophozoites [19,20]. This has been explained as the result of mRNA extraction from cell populations rather than individual cells and by indications that there is a trend, as intra-erythrocytic development progresses, for ‘‘loose’’ multi-var locus transcription to decrease, and a single mRNA type to become predominant, this dominant transcript becoming the sole translated and exported Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) [16,17,21]

Summary

Introduction

Its pathogenesis is closely associated with the adhesive properties of the variable erythrocyte surface antigens, PfEMP1, encoded by the var gene family [1]. These adhesins force erythrocytes infected with the parasite to bind to host receptors such as CD36, CD31/ PECAM1 and CD54/ICAM1 on the endothelial lining of venular capillaries [2,3,4,5]. The more mature, replicating stages of the parasite leave the peripheral circulation, perhaps to avoid elimination by the reticulo-endothelial system of the host spleen Their sequestration damages the host, as occlusion and inflammation around capillaries results in damage to vital organs and sometimes leads to potentially fatal complications such as cerebral malaria [1,6,7]. Switching expression of PfEMP1 antigens with alternative antigenic and cytoadherent properties allows P

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Conclusion