In vitro selection for adhesion of Plasmodium falciparum-infected erythrocytes to ABO antigens does not affect PfEMP1 and RIFIN expression

Abstract

Plasmodium falciparum causes the most severe form of malaria in humans. The adhesion of the infected erythrocytes (IEs) to endothelial receptors (sequestration) and to uninfected erythrocytes (rosetting) are considered major elements in the pathogenesis of the disease. Both sequestration and rosetting appear to involve particular members of several IE variant surface antigens (VSAs) as ligands, interacting with multiple vascular host receptors, including the ABO blood group antigens. In this study, we subjected genetically distinct P. falciparum parasites to in vitro selection for increased IE adhesion to ABO antigens in the absence of potentially confounding receptors. The selection resulted in IEs that adhered stronger to pure ABO antigens, to erythrocytes, and to various human cell lines than their unselected counterparts. However, selection did not result in marked qualitative changes in transcript levels of the genes encoding the best-described VSA families, PfEMP1 and RIFIN. Rather, overall transcription of both gene families tended to decline following selection. Furthermore, selection-induced increases in the adhesion to ABO occurred in the absence of marked changes in immune IgG recognition of IE surface antigens, generally assumed to target mainly VSAs. Our study sheds new light on our understanding of the processes and molecules involved in IE sequestration and rosetting.

Figure 1

Selection for IE adhesion to ABO antigens. Adhesion of erythrocytes infected with late-stage P. falciparum 3D7 to BSA, BSA-H, BSA-A, and BSA-B before (white) and after four rounds of selection on BSA-H (grey), BSA-A (red), and BSA-B (blue), respectively. Adhesion of uninfected erythrocytes to the receptors was always < 5% of the erythrocytes added. Error bars indicate standard deviations of mean results from the number of independent assays indicated in the figure. Statistically significant differences (P < 0.05) of post-hoc pairwise comparisons (Holm-Sidak method) following 1-way ANOVA (P < 0.001) are indicated by lines along the top of the panel.

Figure 2

Formation of rosettes after selection for IE adhesion to ABO antigens. Frequency of rosettes formed by adhesion of uninfected blood group O, A, or AB erythrocytes to erythrocytes infected with late-stage P. falciparum 3D7 (A), FMG (B), or FUP (C) before selection (white) or after selection of IEs for adhesion to BSA-A (red) or BSA-B (blue). Data from one out of two independent experiments with similar results are shown.

Figure 3

Temporal stability of IE adhesion phenotype following selection. Adhesion of P. falciparum 3D7-IEs to BSA (circles) or BSA-A (diamonds) (A), or to BSA-H (triangles) or BSA-B (squares) (B) at various time-points following selection for IE adhesion to BSA-A (red symbols) or BSA-B (blue symbols). Individual data points (symbols), regression lines (heavy lines), and 95% confidence intervals for regression lines (thin lines) are shown.

Figure 4

Adhesion of IEs to cellular receptors. Adhesion of erythrocytes infected with late-stage P. falciparum 3D7 to monolayers of aorta endothelial cells (EC), foreskin EC, BeWo choriocarcinoma cells, or uncoated wells (No cells) before (white) and after four rounds of selection on BSA-A (red) or BSA-B (blue), respectively. Error bars indicate standard deviations of mean results from four (No cells, Aorta EC, Foreskin EC) or three (BeWo cells) independent assays. Statistically significant differences (P < 0. 05) of post-hoc pairwise comparisons (Holm-Sidak method) following 1-way ANOVA (P < 0.01) are indicated by lines along the top of the panel.

Figure 5

Transcription of var genes. Transcription of var genes in ring-stage P. falciparum 3D7 before (left) and after selection × 3 for IE adhesion to BSA-A (centre) or BSA-B (right). Genes are sorted according to structural groups and to the transcript levels of individual genes relative to housekeeping gene in unselected parasites (this order is maintained in all panels). The size of the pies reflect the overall transcript levels relative to housekeeping genes. The var gene pf130003 is identified by an asterisk (*). For additional details regarding individual genes, see Supplementary Data.

Figure 6

Transcription of rif genes. Transcription of rif genes in ring-stage P. falciparum 3D7 before (left) and after selection × 3 for IE adhesion to BSA-A (centre) or BSA-B (right). Genes are sorted according to types (A: red; B: blue; uncharacterized: yellow) and to the transcript levels of individual genes relative to housekeeping gene in unselected parasites. The size of the pies reflect the overall transcript levels relative to housekeeping genes. For details regarding individual genes, see Supplementary Data.

Figure 7

Immune IgG recognition of IEs. Immune IgG recognition of erythrocytes infected with late-stage P. falciparum 3D7 (A), FMG (B), or HB3 (C) before (white) and after four rounds of selection on BSA-A (red) or BSA-B (blue), respectively. Medians (centre lines), central 50% (boxes), central 90% (error bars), and outliers (circles) of levels of IgG in individual plasma samples from 93 Ghanaian children with P. falciparum malaria are shown. Recognition of the IEs by IgG in a pool of plasma from non-exposed donors was always < 5.5 (3D7) or < 1.5 (FMG and HB3).