Abnormal PfEMP1/knob display on Plasmodium falciparum-infected erythrocytes containing hemoglobin variants: fresh insights into malaria pathogenesis and protection

Abstract

Hemoglobin (Hb) variants are associated with reduced risk of life-threatening Plasmodium falciparum malaria syndromes, including cerebral malaria and severe malarial anemia. Despite decades of research, the mechanisms by which common Hb variants - sickle HbS, HbC, α-thalassemia, fetal HbF - protect African children against severe and fatal malaria have not been fully elucidated. In vitro experimental and epidemiological data have long suggested that Hb variants do not confer malaria protection by restricting the growth of parasites in red blood cells (RBCs). Recently, four Hb variants were found to impair cytoadherence, the binding of P. falciparum-infected RBCs (PfRBCs) to microvascular endothelial cells (MVECs), a centrally important event in both parasite survival and malaria pathogenesis in humans. Impaired cytoadherence is associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite's major cytoadherence ligand and virulence factor, on the surface of host RBCs. We propose a model in which Hb variants allow parasites to display relatively low levels of PfEMP1, sufficient for sequestering PfRBCs in microvessels and avoiding their clearance from the bloodstream by the spleen. By preventing the display of high levels of PfEMP1, Hb variants may weaken the binding of PfRBCs to MVECs, compromising their ability to activate endothelium and initiate the downstream microvascular events that drive the pathogenesis of malaria.

Figure 1. The asexual life-cycle of blood-stage Plasmodium falciparum parasites

As early ring-stage parasites (top center) mature, they traffic PfEMP1 and other knob-associated proteins to the surface of their host RBCs, enabling their late ring-stage forms to adhere to microvascular endothelial cells. While sequestered in microvessels, parasites continue to mature into trophozoite and segmented schizonts. Merozoites that egress from schizonts infect RBCs, producing a new brood of ring-stage parasites in the bloodstream. The sequestration of parasitized RBCs leads to multiple deleterious effects in humans. These are initiated by endothelial activation and include the co-sequestration of blood elements (RBCs, leukocytes and platelets), endothelial dysfunction and microvascular obstruction.

Figure 2. Hemoglobin C and sickle hemoglobin S are associated with abnormal PfEMP1/knob display

Scanning (top row) and transmission (bottom row) electron micrographs of P. falciparum-infected red blood cells (RBCs) that are homozygous for normal hemoglobin (Hb) A, or heterozygous for the Hb variants HbC and HbS (sickle-cell trait). Parasitized RBCs (young-ring stage) were obtained from HbAA, HbAC and HbAS Malian children with malaria, immediately cultured ex vivo to the trophozoite stage expressing PfEMP1 and knobs, and then processed for imaging. Blue arrows indicate the normally-shaped, uniformly-distributed knobs characteristic of HbAA RBCs. Red arrows indicate the abnormally-shaped, heterogeneously-distributed knobs observed on significant fractions of HbAC and HbAS RBCs. Scale bars represent 2 μm (top row) and 0.5 μm (bottom row).

Figure 3. Relative cytoadherence of P. falciparum-infected red blood cells to microvascular endothelial cells

Freshly-drawn red blood cells (RBCs) containing the indicated hemoglobin (Hb) variant were inoculated with laboratory-adapted P. falciparum clones or P. falciparum isolates from Malian children with malaria. After one cycle of invasion and development to the trophozoite stage expressing PfEMP1 and knobs, the cytoadherence of these samples was compared to parasitized HbAA samples processed in parallel. In each experiment, the cytoadherence of HbAA samples is normalized to 100%. These summary data are derived from experiments conducted in [30, 31, 54, 58].

Figure 4. A model to explain how hemoglobin variants protect young African children from the severe manifestations of Plasmodium falciparum malaria

In the young infant, fetal hemoglobin (Hb) F works cooperatively with maternally-acquired PfEMP1-specific IgG to effectively prevent the sequestration of P. falciparum-infected red blood cells (PfRBCs), resulting in their clearance from the bloodstream by the spleen. This results in a quiescent endothelium. As the child ages, maternal IgG is catabolized and HbF is quantitatively replaced with normal adult HbAA or one or more Hb variant. In a child with no Hb variants (i.e., HbAA, αα/αα), knobs cover the entire surface of PfRBCs and enable their strong binding to microvascular endothelial cells (MVECs). Endothelial cell activation ensues, characterized in part by increased expression of ICAM-1 and tissue factor. Along with endothelial activation, the co-sequestration of blood elements (RBCs, monocytes and platelets) and activation of the coagulation cascade causes endothelial dysfunction (i.e., vasoconstriction) – shown by narrowing of the microvessel lumen – and microvascular obstruction. This results in a high level of microvascular inflammation, shown by an intense reddening of the endothelial cells. In a child with one or more Hb variant (i.e., HbC, sickle HbS and α-thalassemia), knobs are fewer and mal-distributed on the surface of PfRBCs and thus the number of ligand-receptor interactions is markedly diminished. The weaker binding of PfRBCs to host cells leads to reduced endothelial activation, less co-sequestration of blood elements and preservation of endothelial function. This results in a slightly inflamed endothelium, with sufficient microvascular obstruction to cause uncomplicated malaria, but not enough to cause severe, life-threatening malaria.