Cross-reactive surface epitopes on chondroitin sulfate A-adherent Plasmodium falciparum-infected erythrocytes are associated with transcription of var2csa

Abstract

Malaria in pregnancy is associated with placental accumulation of Plasmodium falciparum-infected erythrocytes (IE) that adhere to chondroitin sulfate A (CSA). Adhesion is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1), a variant parasite protein expressed on the surface of IE and encoded by var genes. Rabbit antiserum was generated against the CSA-adherent P. falciparum line CS2, in which the dominant var transcribed is var2csa, a relatively conserved var gene that has been associated with CSA adhesion. Anti-CS2 recognized genetically distinct CSA-adherent P. falciparum lines but not CD36-adherent parent lines. Reactivity with anti-CS2 correlated with the level of adhesion to CSA. Fluorescence-activated cell sorting according to binding of anti-CS2 showed reactivity was associated with CSA adhesion and transcription of var2csa. These data are consistent with the hypothesis that var2csa encodes a PfEMP1 expressed on the surface of IE, which mediates adhesion to CSA and is relatively conserved between genetically distinct strains of P. falciparum.

FIG. 1.

Summary of P. falciparum lines used in this study. Three genetically distinct laboratory lines, FAF-EA8 (derived by selection of ItG on human umbilical vein endothelial cells [HUVEC]), 3D7, and HM (from a traveler infected in Southeast Asia), were used. (A) FAF-EA8 was selected for adhesion to CSA to generate CS2, selected on HA to generate E8B-HA, and selected on ICAM-1 to generate E8B-ICAM. Rabbit antiserum was generated against the CSA-adherent line CS2 (boldface box). (B) 3D7 was selected on CSA to generate 3C, which in turn was selected on ICAM-1 to produce 3CI and then reselected on CSA to derive 3CIC. Direct selection of 3D7 on ICAM-1 produced 3D7-ICAM. (C) The patient isolate HM was selected on CSA to generate HCS3. The adhesion phenotype of each line is indicated in italics.

FIG. 2.

Rabbit antiserum generated against CS2 trophozoites (anti-CS2) was screened against the CSA-adherent ItG lines CS2 (A) and E8B-HA (B) and the CD36-adherent parent line FAF-EA8 (C) by flow cytometry. Background staining of CS2 with normal rabbit serum (NRS) from an unimmunized rabbit is also shown (A). Binding of malaria-exposed human serum (HS) to FAF-EA8 IE (C) demonstrated the presence of variant surface antigens. The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of each dot plot. The bar graph in each panel shows the adhesion phenotype for each line (mean and standard error of the mean). The percentage of parasitemia (trophozoites) used in each adhesion assay is shown on each graph. Representative results from individual experiments are shown.

FIG. 3.

Anti-CS2 was screened against the 3D7-derived CSA-adherent lines 3C (A) and 3CIC (B) and the CD36-adherent 3D7 line (C) by flow cytometry. Binding of malaria-exposed human serum (HS) to 3D7 IE (C) indicated the presence of variant surface antigens. The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of each dot plot. The bar graph in each panel shows the adhesion phenotype for each line (mean and standard error of the mean). The percentage of parasitemia (trophozoites) used in each adhesion assay is shown on each graph. Representative results from individual experiments are shown.

FIG. 4.

Anti-CS2 was screened against the CSA-selected line HCS3 (A) and the Southeast Asian isolate from which it was derived, HM (B), by flow cytometry. Binding of malaria-exposed human serum (HS) to HM IE (B) confirmed the presence of variant surface antigens. The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of each dot plot. The bar graphs in panels A and B show the adhesion phenotype for each line (mean and standard error of the mean). The percentage of trophozoites used in each adhesion assay is shown on each graph. Representative results from individual experiments are shown. (C) Northern blots of HM and HCS3, obtained using probes against the conserved var exon 2 (which should detect all var genes) and var2csa.

FIG. 5.

E8B-HA IE with mixed adhesion phenotypes after several weeks in culture (A) were reselected on CD36 (B), CSA (C), or HA (D), and their recognition by anti-CS2 was determined. The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of each dot plot. The bar graph in each panel shows the adhesion phenotype for each line (mean and standard error of the mean). The percentage of trophozoites used in adhesion assays is shown on each graph. Representative results from individual experiments are shown.

FIG. 6.

E8B-HA IE with mixed phenotypes after several weeks in culture (A) were cell sorted according to recognition by anti-CS2. Anti-CS2-positive IE (B) and anti-CS2-negative IE (C) were grown for approximately 2 weeks, and the adhesion phenotypes were determined. The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of each dot plot. Binding of malaria-exposed human serum (HS) to anti-CS2 negative IE (C) demonstrated the presence of variant surface antigens. The bar graph in each panel shows the adhesion phenotype for each line (mean and standard error of the mean). The percentage of trophozoites used in each adhesion assay is shown on each graph. Representative results from individual experiments are shown.

FIG. 7.

(A) Northern blot of ring-stage RNA from E8B-HA sorted lines (anti-CS2 pos, anti-CS2 neg) and selected lines (sel CSA, sel CD36, sel HA [see Fig. 5 and 6]) using a var exon 2 probe (which should detect all var genes) and a var2csa-specific probe. The dominant var2csa transcript in anti-CS2-positive, CSA-selected (sel CSA), and HA-selected (sel HA) lines is indicated (arrow). (B) Quantitative RT-PCR using var2csa-specific primers with E8B-HA sorted and selected lines (means and standard deviations).

FIG. 8.

Anti-CS2 was screened against the 3D7 lines selected for adhesion to ICAM-1, 3CI (A) and 3D7-ICAM (B). The percentage of trophozoites bound by anti-CS2 is indicated in the upper right quadrant of each dot plot. The bar graphs in panels A, B, and D show the adhesion phenotype for each line (mean and standard error of the mean). The percentage of trophozoites used in each adhesion assay is shown on each graph. Representative results from individual experiments are shown. (C) Northern blot of ring-stage RNA from 3D7-ICAM, 3CI, and HCS3 (included as a positive control) using a var exon 2 probe (which should detect all var genes) and a var2csa-specific probe. (D) Anti-CS2 screened against the genetically distinct ICAM-1-adherent line E8B-ICAM.