A single member of the Plasmodium falciparum var multigene family determines cytoadhesion to the placental receptor chondroitin sulphate A

Abstract

In high-transmission regions, protective clinical immunity to Plasmodium falciparum develops during the early years of life, limiting serious complications of malaria in young children. Pregnant women are an exception and are especially susceptible to severe P. falciparum infections resulting from the massive adhesion of parasitized erythrocytes to chondroitin sulphate A (CSA) present on placental syncytiotrophoblasts. Epidemiological studies strongly support the feasibility of an intervention strategy to protect pregnant women from disease. However, different parasite molecules have been associated with adhesion to CSA. In this work, we show that disruption of the var2csa gene of P. falciparum results in the inability of parasites to recover the CSA-binding phenotype. This gene is a member of the var multigene family and was previously shown to be composed of domains that mediate binding to CSA. Our results show the central role of var2CSA in CSA adhesion and support var2CSA as a leading vaccine candidate aimed at protecting pregnant women and their fetuses.

Figure 1

Targeted gene disruption of Plasmodium falciparum var2csa. (A) Schematic representation of the disruption of var2csa by double-crossover integration. The pHTK-var2csa plasmid contains the thymidine kinase gene, hDHFR, and the sequences corresponding to DBL3-X and DBL5-ɛ of var2csa. The DBL4-ɛ region has been deleted and replaced by the hDHFR gene. The different Duffy binding-like (DBL) domains and the transmembrane (TM) domain, and carboxy-terminal cytoplasmic domain (ATS) of var2csa are shown. Homologous target sequences are shown in dark grey. Sizes of DNA fragments are shown in kilobases (kb). Restriction enzyme sites and the expected restriction fragments are indicated. Hybridization probes are indicated as black bars. (B) Knockout of var2csa by a double-crossover event. Southern blot analysis of genomic DNA from representative mutant clones 1F1 and 2A5 and the parental FCR3 strain using BamHI, EcoRV and PvuII restriction enzymes. Hybridization was carried out with DBL3-X- and DBL5-ɛ-specific probes. The positions of the probes are shown in (A). (C) Insertion of pHTK-var2csa in chromosome 12. Southern blot analysis of chromosomal DNA derived from FCR3 wt (wild type) and the representative disrupted mutant clones 1F1 and 2A5. Chromosomes were separated by pulsed-field gel electrophoresis, then transferred onto a nylon membrane and hybridized with probes specific for DBL5-ɛ, hDHFR and clathrin heavy chain. The position of chromosome 12 is indicated.

Figure 2

FCR3Δvar2csa clones cytoadhere to CD36 and express a var gene that is different from var2csa. (A) Cytoadhesion of the var2csa disruption mutants to receptors coated to plastic Petri dishes. Erythrocytes infected with the Plasmodium falciparum FCR3-CSA, FCR3-CD36 and the FCR3Δvar2csa clones 1F1 and 2A5 were analysed for cytoadhesion to CSA and CD36. Data are the mean±s.e.m. of IE per square millimetre (IE/mm²) adhering to CSA-coated (left panel) and CD36-coated (right panel) plastic Petri dishes, as determined in three independent assays. (B) Northern analyses of total RNA isolated from ring-stage (R) and trophozoite-stage parasites (T) FCR3-CSA, FCR3-CD36 and the representative FCR3Δvar2csa clones 1F1 and 2A5. The membrane was hybridized with a probe specific for var2csa DBL3-X and semiconserved varT11.1 exon II.

Figure 3

FCR3Δvar2csa mutants show no chondroitin sulphate A (CSA)-specific cytoadhesion after selection on CSA-expressing cell lines and recombinant human thrombomodulin. Mean±s.d. of IE adhering per square millimetre (IE/mm²) for four different fields is shown (A,B). (A) Selection of FCR3Δvar2csa mutants and parental FCR3 parasites on recombinant human thrombomodulin. Erythrocytes infected with FCR3-CSA, FCR3-CD36, 1F1 and 2A5 were selected four times on recombinant human thrombomodulin-CSA coated to Petri dishes. (B) Selection of FCR3Δvar2csa mutants and parental FCR3 parasites on Sc1707. Trophozoite-stage Plasmodium falciparum clones FCR3-CSA, FCR3-CD36, 1F1 and 2A5 were subjected to repeated rounds of selection over Sc1707 cells, followed by evaluation of adhesion to Sc1707. (C,D) Adhesion profiles of P. falciparum IE after selection on Sc1707 cells. Trophozoite-stage P. falciparum parasites FCR3-CSA, FCR3-CD36, 1F1 and 2A5 were subjected to repeated rounds of selection over Sc1707 cells, followed by evaluation of adhesion to CSA-coated (C) and CD36-coated (D) plastic Petri dishes. Adhesion after selection is shown for FCR3-CSA¹⁷⁰⁷, FCR3-CD36¹⁷⁰⁷, 1F1¹⁷⁰⁷ and 2A5¹⁷⁰⁷. Data are the mean±s.e.m. of IE per square millimetre adhering to CSA- and CD36-coated plastic Petri dishes, as determined in three independent assays.