The 3D7var5.2 (varCOMMON) type var gene family is commonly expressed in non-placental Plasmodium falciparum malaria☆
Introduction
The display of variable adhesion antigens at the surface of the Plasmodium falciparum infected erythrocyte (IE) is a characteristic feature of erythrocytes infected by mature blood stages and it is critical for the survival and transmission of the parasite. In persistent infections the parasite evades the mounting immune response by antigenic variation leading to characteristic waves of parasitemia [1], [2]. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is the most prominent of the variable antigens exported to the erythrocyte surface. It mediates the sequestration of IE in the micro-vasculature of the brain, the placenta and other organs by adhering to host receptors on the endothelium (cytoadhesion) and on uninfected erythrocytes (rosetting). Sexual forms (gametocytes) also express PfEMP1 in the early, sequestering stages but not in the later circulating forms [3].
PfEMP1 proteins are encoded by a large and diverse family of var genes, which typically consist of a highly variable 5′ exon encoding the extracellular domain, a relatively conserved intron and a conserved 3′ exon encoding an internally located acidic terminal segment [4]. It has previously been suggested that each parasite contains approximately 50 var genes per haploid genome [5], [6], [7], [8], [18], an estimate recently confirmed with the elucidation of the genome sequence of 3D7 parasites revealing 59 var genes each encoding a unique PfEMP1. Additionally, 3D7 parasites contain several pseudo- and truncated var genes [9].
Most var genes are located in the subtelomeric region of P. falciparum chromosomes [10], [11], [12]. Frequent ectopic recombination events in this chromosomal area may generate a high degree of diversity in the total var gene pool [13], [14], [15], [16]. In the course of a persistent infection the parasite evades the host immune attack by switching the expressed var gene which is paralleled by a change of the antigenic and adhesive phenotype of the IE [17]. While a number of var genes are transcribed in ring stage parasites, only one gene was found expressed in the trophozoite suggesting a form of allelic exclusion [8], [18], [19]. The mechanism by which the parasite selectively activates a particular var gene while silencing others is not yet understood. Recently, Deitsch et al. [20] identified upstream and in the intron sequences of var genes that cooperatively act as regulatory sequence elements involved in the silencing of var gene transcription [20]. However, not all var genes appear to contain these elements, which may indicate that transcription of a subset of var genes is regulated by alternative means.
The expression of var genes has been extensively studied in laboratory strains. By contrast, little is known about var gene expression in malaria patients. In this study, we therefore investigated var gene expression in clinical isolates from children in Gabon with mild malaria. Unexpectedly, the isolates commonly expressed a highly conserved var gene sequence, here referred to as varDBL1-COMMON. We found this sequence also transcribed in a novel placenta-binding laboratory clone 3D7S8, here as part of a large, 9 domain var gene in 3D7 parasites, var5.2. Recent studies on var gene expression in placental isolates identified two commonly expressed families of DBLγ containing genes [22], [23]. These two families were defined by Rowe et al. [23] on the basis of their homology to the 5′-end of either the var5.2 of 3D7 parasites or to the previously characterised chondroitin sulfate A (CSA)-binding var gene FCR3varCSA [21]. Consequently, the data suggested that an effective placental malaria vaccine based on the DBLγ domain would have to target only a limited number of var genes. By contrast, our data presented here show that the expression of a var gene family highly homologous to the 5′-end of 3D7var5.2 is not restricted to placental malaria. As this var type is commonly transcribed in the field we propose that it be referred to as varCOMMON.
varCOMMON is structurally related to FCR3varCSA in that both gene types contain a similar array of seven DBL domains. Intriguingly, while being clearly distinct in the 5′ region, varCOMMON and FCR3varCSA are highly homologous in the 3′-end of exon1 comprising the DBL5 to DBL7 encoding region [23]. It is important to note, however, that the varCOMMON gene, in contrast to FCR3varCSA, lacks a common intron and an exon2 domain. Our data suggest that varCOMMON related genes are commonly transcribed and highly conserved genes, which are not exposed on the IE surface. Furthermore, our results indicate that 3D7S8 mature stage trophozoites transcribe at least two var genes, varCOMMON and a second yet to be elucidated PfEMP1 type, which is expressed at the IE surface and may facilitate placenta binding of 3D7S8 parasites by novel mechanisms.
Section snippets
Parasites
3D7S8 was sub-cloned by micromanipulation from 3D7AH1 parasites, a clone previously obtained by limiting dilution from a patient isolate NF54. FCR3S1.2 is a sub-clone obtained previously by micromanipulation from the clone FCR3S1 [24]. Peripheral blood samples from randomly selected children with malaria were obtained in 1995 and 1996 at the Albert Schweitzer Hospital in Lambarene, Gabon [25], [26], an area hyperendemic for malaria [27]. The genetic analysis of parasites was approved by the
A 3D7var5.2 DBL1α sequence, varDBL1-COMMON is commonly expressed in field isolates2
In order to analyse the var gene repertoire expressed in field isolates 23 frozen blood samples of Gabonese children with mild malaria symptoms were thawed and in vitro cultured. Within a 72 h period sixteen of the isolates developed into mature trophozoites, which were isolated using Percoll gradient centrifugation and extracted for RNA and DNA. We amplified var gene messenger RNA using reverse transcription and polymerase chain reaction (RT-PCR) with universal var primers, as recently
Discussion
The variation in antigenicity of the IE surface, the switch of PfEMP1 variants, provides a logical framework of how the parasite evades immune clearance while retaining the primary function of adhesion. Indeed, studies have shown polymorphism of the var gene repertoire in natural parasite populations in which the sequence variability was found to be similar within and between isolates [6], [7], [15], [16], [44], [45]. In this context, our observation of a highly conserved var-sequence expressed
Acknowledgements
This work was funded by grants from the European Union (QLRT-PL-1999-30109 and QLK2-1999-01293 (Euromalvac I)), and the Swedish Research Council. Gerhard Winter was supported by the Wenner-Grenska Foundation. Kirsten Flick was recipient of a Deutsche Forschungsgemeinschaft stipend (grant Fl328/1-1).
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Note: Nucleotide sequence data are available in the GenBank™, EMBL and DDBJ databases under accession numbers AF528110-AF528155 and AF528100-AF528109.
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Present address: Canadian Science Center for Human and Animal Health, Special Pathogens Program, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E3R2.