VAR2CSA is the principal ligand for chondroitin sulfate A in two allogeneic isolates of Plasmodium falciparum
Introduction
In areas of endemic transmission the burden of malarial disease falls primarily on children and pregnant women. Malaria in pregnancy causes mortality and morbidity principally through maternal anemia and low birth weight [1]. Susceptibility during pregnancy of previously immune women is associated with sequestration within the placenta of parasitised red blood cells (pRBCs) that express unique variant surface antigens (VSAs) [2]. The restriction of these VSAs to pregnant women correlates with the restricted adhesion phenotype observed in placental parasites. The host receptors implicated in adhesion of pRBCs to placenta are CSA, hyaluronic acid and non-immune globulins [3], [4], [5] whereas other host receptors, including CD36 and ICAM-1, mediate sequestration of pRBCs in non-pregnant individuals. Presumably the placenta selects for parasites that bind CSA by providing a novel host receptor in a form of CSA that is absent or not accessible to pRBCs in non-pregnant individuals. Because pRBCs expressing the CSA adhesion ligand are not selected for in non-pregnant individuals [6], malaria-exposed women do not possess immunity to pRBCs expressing the CSA adhesion ligand prior to pregnancy.
Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) molecules are expressed on the surface of the pRBCs and mediate adhesion of the pRBCs to various host receptors. Each of the approximately 60 members of the var multigene family present within a single parasite encodes a different PfEMP1 [7]. var genes are subject to transcriptional control such that a single full-length var gene transcript is dominant at one time within a parasite [8], [9]. Switching between the expressed var genes allows parasites to evade the immune response through antigenic variation and also to change the adhesion phenotype of the pRBC.
Various studies have proposed different PfEMP1 molecules as the CSA adhesion ligands important in pathogenesis of malaria in pregnancy, as reviewed elsewhere [10]. However, the most consistent data implicates the PfEMP1 molecule VAR2CSA [11]. Transcription of var2csa is associated with adhesion to CSA and hyaluronic acid [11], [12] and also with reactivity to serum raised against CSA binding pRBCs in allogeneic parasite lines [13]. Recombinant domains of var2csa bind to CSA [14] and react with sera from residents of endemic areas in a gender-specific, parity-dependent fashion [15]. Recent evidence that VAR2CSA was exclusively responsible for CSA adhesion in a single parasite line [16] corroborated these data but it is possible that the parasite line studied lacked CSA binding PfEMP1(s) present in other parasites because of the tremendous diversity in the genomic var repertoire between allogeneic parasites. We disrupted var2csa in two allogeneic CSA binding parasite lines to determine whether other PfEMP1 in either isolate could bind CSA. Adhesion to bovine trachea CSA was ablated by var2csa deletion but was recovered in one mutant parasite that transcribed at least two other var genes. However, the mutant parasites did not react with sera in a gender-specific, parity-dependent fashion, suggesting that most malaria in pregnancy arises from parasite adhesion to CSA in association with expression of the unique epitopes of VAR2CSA.
Section snippets
Results and discussion
To determine the importance of var2csa during malaria in pregnancy we disrupted var2csa in both P. falciparum 3D7 derived 3C parasites, that are isogenic with NF54 parasites, and ItG derived CS2 parasites. Both the 3C and CS2 parasites were repeatedly selected for a high level of adhesion to CSA (Table 1) and transcribed predominantly var2csa [12]. Both CS2 parasites and var2csa transcribing NF54 parasites are recognized by sera from residents of malaria endemic areas in a gender-specific and
Plasmid construction
Two fragments from within the var2csa gene were amplified from both CS2 and 3C parasite DNA and cloned into the plasmid transfection vector pHHT-TK [18]. The first fragment spanning nucleotides 1395–2214 of var2csa (ItG) was amplified from CS2 DNA using the primers 5′-TGccgcggTGTAAAGTTGGGTGTTCGTGAAA and 5′-actagtACCATCAGCATTACACGTAGTAATATTCAT. The second fragment spanning nucleotides 2960–3740 of var2csa (ItG) was amplified using the primers 5′-atcgatGTGGTAGCGCACGAACTATGAA and
Acknowledgements
The authors thank James Beeson and Rintis Noviyanti for advice and assistance. This work was funded by the National Health Medical Research Council of Australia. Stephen J. Rogerson is a Wellcome Trust Senior Fellow (063215).
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