Plasmodium falciparum associated with severe childhood malaria preferentially expresses PfEMP1 encoded by group A var genes

Abstract

Parasite-encoded variant surface antigens (VSAs) like the var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family are responsible for antigenic variation and infected red blood cell (RBC) cytoadhesion in P. falciparum malaria. Parasites causing severe malaria in nonimmune patients tend to express a restricted subset of VSA (VSA(SM)) that differs from VSA associated with uncomplicated malaria and asymptomatic infection (VSA(UM)). We compared var gene transcription in unselected P. falciparum clone 3D7 expressing VSA(UM) to in vitro-selected sublines expressing VSA(SM) to identify PfEMP1 responsible for the VSA(SM) phenotype. Expression of VSA(SM) was accompanied by up-regulation of Group A var genes. The most prominently up-regulated Group A gene (PFD1235w/MAL7P1.1) was translated into a protein expressed on the infected RBC surface. The proteins encoded by Group A var genes, such as PFD1235w/MAL7P1.1, appear to be involved in the pathogenesis of severe disease and are thus attractive candidates for a vaccine against life-threatening P. falciparum malaria.

Figure 1.

Fold changes in var gene transcription by late-stage (36–48 h after invasion) P. falciparum 3D7 before and after antibody selection of parasites for selection of VSA_SM-type antigens using DynaBeads coated with IgG from three different pools of plasma from semi-immune African children (SM1, SM2, and SM3; a–c) and for adhesion to TrHBMEC (d; see Materials and Methods for details). Transcription levels were measured using real-time PCR and primers specific for 59 var genes and 1 pseudogene (reference and Table S1). Experiments with SM1 were repeated five times and results are shown as means ± SD. Solid bars indicate highly transcribed var genes. These genes had the 15 lowest Ct values in selected and/or unselected 3D7. A threefold change in var gene transcription (dashed lines) was arbitrarily defined as the cut off for biologically significant changes in var gene transcription. Primer identity and calculation of selection-induced var gene transcription is described in Materials and Methods and reference . The grouping of var genes is described in reference .

Figure 2.

Changes in var gene transcription in synchronized ring-stage (30 h) P. falciparum 3D7 after antibody selection with the SM1 and SM2 pools (a and b) or selection for adhesion to TrHBMEC (c) as described in the legend for Fig. 1. A fold-change of two (dotted lines) was defined as the cut off for biological interesting changes in var gene transcription.

Figure 3.

Relative levels of var transcripts in 10 μg total RNA obtained from unselected (1) and antibody-selected 3D7 (2) ring-stage (30 h)–infected RBCs. Northern blots were probed with DIG-labeled RNA probes targeting PFD1235w/MAL7P1.1 (a), PF08_0107 (b), and MAL6P.314 (c). Transcript sizes were 12.5 (PFD1235w/MAL7P1.1), 8.3 (PF08_0107), and 5.7 kb (MAL6P.314). An ethidium bromide–stained gel is shown to allow comparison of total RNA amounts (d).

Figure 4.

Expression of PfEMP1 in unselected (a, c, and e), antibody-selected (b and d), and TrHBMEC-selected (f) trophozoite/schizont stage 3D7. Samples were run in 5% SDS-PAGE gels, blotted, and probed with antiserum against a conserved part of the intracellular acidic segment ATS (a and b) or against DBL5-δ of PFD1235w/MAL7P1.1 (c–i). Antibody-selected parasites expressed PFD1235w/MAL7P1.1 (***; b and d) and a PfEMP1 with mol wt corresponding to the predicted mol wt of MAL6P1.316 (**; b). Both selected and unselected parasites expressed a PfEMP1 with mol wt corresponding to the predicted mol wt of PF08_0107 (*; a and b). TrHBMEC-selected 3D7 expressed PFD1235w/MAL7P1.1 (f). Western blots of SDS-extracted RBCs infected by NF54 obtained from a Dutch volunteer (reference 27) on days 8, 9, and 10 of an experimental, mosquito-induced infection (g–i) probed with the DBL5-δ antibody revealed expression of PFD1235w/MAL7P1.1 on day 10 (i). Surface expression of antigens in P. falciparum 3D7 before (a1–a4) and after (b1–b4) antibody selection. Murine plasma antiserum against recombinant DBL5-δ of PFD1235w/MAL7P1.1 protein reacted with the surface of most antibody-selected 3D7-infected RBCs in FACS^® (b1, green line). This reaction was abolished by trypsin treatment (b1, purple line). In contrast, the antiserum only reacted with a minority of unselected 3D7 (a1, green line). Reactivity with pre-vaccination mouse serum is shown for comparison (shaded histograms). VSA-specific IgG reactivity in plasma from a semi-immune African child (a2 and b2, red line) and a clinically immune African adult (a2 and b2, blue line) confirmed the VSA_UM phenotype of unselected 3D7 and the VSA_SM phenotype of antibody-selected 3D7. Localization of the PFD1235w/MAL7P1.1 protein using confocal microscopy and murine plasma anti–DBL5-δ antibodies (a3, a4, b3, and b4). Ethidium bromide staining of DNA in the nuclei is red/orange and staining of PFD1235w/MAL7P1.1 protein using FITC-labeled antibodies is green. Pre-vaccination mouse plasma did not stain infected RBCs (not depicted).

Figure 5.

Plasma antibody levels to recombinant DBL5-δ (a) and CIDR1-α (b) domains of the PFD1235w/MAL7P1.1 protein in Tanzanian children and adults, and in Danish donors without P. falciparum exposure (DK). For competition ELISA experiments, plates were coated with recombinant CIDR1-α domains of the proteins encoded by PFD1235w/MAL7P1.1 (c) and PFE1640w/var1 (d), and ELISA reactivity was measured in three different plasma samples, which had been preincubated with increasing concentrations of homologous or heterologous fusion protein as indicated in the top part of each panel. Nondepleted (ND) plasma was included for comparison. Sequence similarity between PFD1235w/MAL7P1.1 and other genes of the var4 family were identified in field isolates. Schematic domain structure of PFD1235w/MAL7P1.1 (var4; e). The line indicates the position of the 2.3-kb fragment (35 Ext) sequenced and numbered line (35) position of the primer pair used for real-time PCR (refer to Figs. 1 and 2, and Table I). Sequence alignment of PFD1235w/MAL7P1.1, BM021, and BM048 (f). The sequences shown correspond to line 35 Ext in (e). These sequence data are available from GenBank/EMBL/DDBJ under accession nos. AY584238 and AY584239.