Multiple var gene transcripts are expressed in Plasmodium falciparum infected erythrocytes selected for adhesion

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Abstract

Adherence of Plasmodium falciparum-infected erythrocytes to the post-capillary endothelium is an important characteristic of malaria infection. The adhesion is mediated predominantly by P. falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a clonally variant protein expressed on the surface of infected red blood cells that appears to be a target of protective immunity. A multi-membered var gene family encodes PfEMP1 and switching expression of different var genes conveys different antigenic and adhesive properties to infected red blood cells. Knowledge about transcriptional control of phenotypic expression, or the mechanisms that allow multiple binding specificities, is very limited. Here, we describe a series of phenotypic selection experiments, which resulted in the expression of different PfEMP1 and the detection of multiple full-length var gene transcripts in the mature trophozoite stage. However, a dominant form of PfEMP1 appeared to be expressed, which suggested that most var transcripts do not lead to a surface expressed PfEMP1 molecule. Parasites bound to specific receptors still expressed multiple full-length var genes and mature trophozoites selected for increased adhesion to a specific receptor retained the ability to bind to multiple receptors. Our findings suggest that a defined adhesive phenotype can be associated with expression of multiple var genes.

Introduction

Mature parasites are rarely seen in the peripheral circulation of the host during Plasmodium falciparum infection, as the parasitised erythrocytes adhere to the microvascular endothelium and sequester in a variety of organs, thus evading splenic clearance. This parasite strategy is not without consequence for the host, and sequestration in specific organs, such as the brain [1] or the placenta [2], [3] is an important pathophysiological correlate with outcome of infection. Many cell adhesion molecules have now been reported in the literature, including CD36 [4], [5], intercellular adhesion molecule-1 (ICAM-1) [6], chondroitin sulfate A (CSA) [7] vascular cell adhesion molecule-1, E-selectin [8], platelet-endothelial cell-adhesion molecule #1(PECAM−1/CD31) [9], P-selectin [10], and hyaluronic acid (HA) [11]. Four of these receptors in particular have attracted attention, because of their association with clinical malaria, ICAM-1 is a candidate for parasite adhesion in cerebral malaria [12]; CSA and HA for adherence of parasitised erythrocytes to syncytiotrophoblasts in placental sequestration [11], [13], and CD36 adhesion is a trait common to many clinical isolates [14], [15].

The parasite derived protein P. falciparum erythrocyte membrane protein-1 (PfEMP-1) is expressed on the surface of erythrocytes infected with mature parasites [16] and has been directly implicated in the adhesion of parasitised erythrocytes to a number of receptors, including CD36, ICAM-1, thrombospondin [17], CSA [18], [19], as well as red blood cells to form rosettes [20], [21]. A large and diverse range of antigenically variant types of PfEMP1 appears to exist in the natural parasite population [14], [22] and PfEMP1 is known to be clonally variant [23], [24]. As antigenic type and adhesion phenotype is co-modulated, alteration of binding to a host receptor may be associated with a change of antigenic type [25], [26]. Antibody against a spectrum of variant antigenic types of this parasite molecule is one of the few correlates with acquired immunity against malaria [27], [28], suggesting that parasite survival and pathogenesis depend upon a critical balance between expression of functional binding regions and evasion of antibody response [29].

PfEMP1 is encoded by the extensive and diverse var gene family (up to 50 members per genome, [24], [30], [31]. The genes can be found on all chromosomes of the parasite and located in central and sub-telomeric positions [32], but little is known about the mechanisms of switching and selective expression of var genes, which produce the different forms of PfEMP1 and confer on the parasitised erythrocyte the ability to adhere to specific receptors. It has been shown that individual parasitised cells can bind to multiple receptors [33], [34], but it is not clear whether a single cell can express multiple PfEMP1. Recent studies have suggested that a single PfEMP1 can mediate binding to both CD36 and ICAM-1 through different domains of the molecule [35], and an expressed portion of PfEMP1 could mediate binding to various host receptors, including platelet–endothelial cell adhesion molecule 1 (PECAM-1/CD31), the blood group A antigen, normal immunoglobulin M, three virulence-associated receptor proteins, a heparan sulfate-like glucosaminoglycan, and CD36 [36].

A mechanism of var gene transcriptional switching was proposed from earlier studies using the FCR3 isolate in which multiple var genes are transcribed during the immature ring stage, but only a single gene conferring adhesive phenotype is detected in the mature trophozoite-stage parasite [37], [38]. It has also been shown that upon selection for adherence to a specific receptor, a parasite population reproducibly transcribes a distinct var gene [38]. In another study, although multiple var genes were detected in ring stage parasites by reverse transcriptase polymerase chain reaction (RT-PCR), only one full-length transcript was detected by Northern blotting, suggesting that a single dominant var gene type was expressed [39]. These authors hypothesised that most ring stage transcripts are degraded before being translated into protein or are present at low levels of sub-populations of full-length transcripts, which are not detectable by Northern analysis.

In order to further investigate the relationship between var gene expression and the adherence of parasitised erythrocytes to specific receptors, the parasite line 3D7 was serially selected in vitro for adhesion to CSA and ICAM-1, then selected back to CSA. Analysis of var gene transcription in the series of phenotypically characterised parasite populations showed that each selected line expressed several var transcripts at the trophozoite stage. Analysis of synchronised trophozoites bound to CSA indicated that multiple var genes can be expressed in a phenotypically homogeneous population of mature parasites.

Section snippets

Parasites

P. falciparum selected lines were derived from 3D7 (a gift from David Walliker, University of Edinburgh) and cultivated in vitro by standard methods [40] in a gas mixture of 5% CO2, 1% O2 in N2, in medium composed of RPMI 1640 (MultiCel, Trace Scientific Ltd. Melbourne, Australia), buffered with HEPES (Gibco, BRL), supplemented with hypoxanthine (50 μg ml−1; Calbiochem, La Jolla, CA), gentamicin (2.5 μg ml−1), and sodium bicarbonate (25 mM). Parasites were cultured in 3–5% haematocrit of blood

Parasite selection results in switching of predominant binding phenotype with retention of binding to CD36

In order to study phenotype switching and its association with PfEMP1 and var gene expression, the 3D7 parasite line was selected for binding to CSA then cloned to give rise to 3C (Fig. 2). Selection of 3C to ICAM-1 was subsequently performed to produce 3CI. Selection back from 3CI to CSA binding gave rise to 3CIC and parasites were again cloned.

The binding characteristics of 3D7 selected lines are shown in Table 2. The parental 3D7 line exhibited binding to CD36, but virtually no adhesion to

Discussion

Despite selection on CSA or ICAM-1, and cloning by limiting dilution, all of the lines derived from 3D7 could bind to multiple receptors with the common phenotypic characteristic of binding to CD36. Western blot analysis of proteins from the mature trophozoites detected changes in the dominant PfEMP1 protein (or proteins) expressed in each selected parasite.

The ability of all selected parasite lines to bind to CD36 has a number of possible explanations. It is possible that every expressed

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia. The authors thank James Beeson for critically reading and reviewing the manuscript; Heather Saunders for final typing of the manuscript; Jenny Thompson, for YAC screening; Kathy Davern, Anne Thaus, Tim Byrne for technical assistance; Natalie Roberts for binding data analysis; Rob Good for advice on analysis of multiple var gene transcripts. Thanks also go to The Malaria Genome Project. Human red blood

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    Note: Nucleotide sequences reported in this paper are available at the GenBank™ database and have accession numbers (AF306395-AF306418).

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