VAR2CSA is the principal ligand for chondroitin sulfate A in two allogeneic isolates of Plasmodium falciparum

https://doi.org/10.1016/j.molbiopara.2006.03.006Get rights and content

Abstract

Malaria during pregnancy causes serious disease that is associated with sequestration in the placenta of Plasmodium falciparum infected erythrocytes that adhere to several host receptors, including chondroitin sulfate A (CSA). The principal CSA binding ligand associated with placental sequestration is the P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var2csa gene. We disrupted the var2csa gene in two allogeneic parasites and ablated CSA binding. However, in one parasite line we were able to re-select for adhesion to bovine trachea CSA associated with transcription of two var genes, var-CS2 and varP. Parasites transcribing parts of var-CS2 and varP were present in the placentae of some infected women but the mutant parasites that transcribed var-CS2 and varP were recognized by sera from men and pregnant women independent of parity. This work raises the possibility that the PfEMP1 molecules encoded by var-CS2 and varP may be minor contributors to placental malaria but also confirms the importance of the immunodominant, conserved var2csa PfEMP1s in pregnancy associated malaria and strengthens the case for var2csa as a pregnancy-specific malaria vaccine.

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).

References (32)

  • S.C. Chaiyaroj et al.

    Cytoadherence characteristics of Plasmodium falciparum isolates from Thailand: evidence for chondroitin sulfate A as a cytoadherence receptor

    Am J Trop Med Hyg

    (1996)
  • M.J. Gardner et al.

    Genome sequence of the human malaria parasite Plasmodium falciparum

    Nature

    (2002)
  • Q. Chen et al.

    Developmental selection of var gene expression in Plasmodium falciparum

    Nature

    (1998)
  • J.A. Rowe et al.

    The role of Plasmodium falciparum var genes in malaria in pregnancy

    Mol Microbiol

    (2004)
  • A. Salanti et al.

    Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria

    Mol Microbiol

    (2003)
  • M.F. Duffy et al.

    Broad analysis reveals a consistent pattern of var gene transcription in Plasmodium falciparum repeatedly selected for a defined adhesion phenotype

    Mol Microbiol

    (2005)
  • Cited by (97)

    • Biosensors for Detection of Human Placental Pathologies: A Review of Emerging Technologies and Current Trends

      2019, Translational Research
      Citation Excerpt :

      The interface between maternal blood and villi interior is “multinuclear giant cell” syncytiotrophoblast where nearly the entire maternofetal exchange takes place,319 as well as infected erythrocytes sequester during PM.320 It is assumed at present that only one member of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of adhesins, VAR2CSA, which binds to placental receptor chondroitin sulfate A (CSA) that expresses on the surface of syncytiotrophoblast, is playing a crucial role in PM.321-323 Several studies clearly demonstrated that cytoadhesion directly affects vascular inflammation.324,325

    • Knobs, Adhesion, and Severe Falciparum Malaria

      2023, Tropical Medicine and Infectious Disease
    View all citing articles on Scopus
    View full text