Pursuing the fever trail : Pathogenesis of blood-stage P. falciparum malaria & pregnancy

Abstract: The burden of the most virulent form of human malarias, Plasmodium falciparum, is concentrated among young children and pregnant women in sub-Saharan Africa. Excessive sequestration of parasite-infected red blood cells (pRBCs) in the capillary beds of different organs is the hallmark of P. falciparum infection contributing to pathology and severe outcomes. Adhesive interactions between parasite proteins at the pRBC surface and host receptors on vascular cell-linings or on immune cells in thought essential in this process. P. falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the highly polymorphic var multi-gene family, is the principal virulence factor involved in both cytoadhesion and antigenic variation. The main aim of this thesis is to enhance our knowledge on both of these processes in relation to severe disease, with a special focus on pregnancy-associated malaria. Histological evidence of placental P. falciparum infection was observed in 13.9% of the delivering mothers at Mulago hospital s labour suite, in Kampala Uganda. Placental infection was associated with parity (P=0.039) and the main burden was concentrated among gravidae 1 to 3. Infection was also associated with adverse outcomes for the mother and the newborn baby with 3.3 times increased risk of maternal anaemia in all gravidities (OR: 3.3; CI: 1.6-6.9) and 200-300 grams of reduced birthweight (P=0.031) (Paper I). PRBCs eluted from infected placentas of a representative sub-fraction of the cases demonstrated adhesive capacity for a number of host receptors: chondroitin sulphate A (CSA), Hyaluronic acid (HA) and non-immune immunoglobulins (Igs). A majority of the isolates had a capacity to adhere to all three receptors (47%), whilst the remainder interacted with different combination of two or a single receptor. The data also implied that the same parasite subpopulation in each isolate co-binds CSA and Igs. The observed link between the two phenotypes was further established in a panel of laboratory isolates of diverse geographical backgrounds. The PfEMP1 variant, VAR2CSA, was dominantly expressed in the Ig-CSA binding isolates. Employing CHO-cells stably transfected with the six different domains of VAR2CSA, Ig-binding was mapped to three out of the six domains. Two of the domains had previously been reported to bind CSA. The same ligand may thus be involved in both adhesive events, which may explain the observation made in placental isolates (Paper II). By switching expression to new PfEMP1 variants the parasite alters its adhesive signature but at the same time evades the evolving immune responses. To understand the dynamics behind switching, var gene expression was systematically assessed over time. The parasites eventually converged to transcribe the same var gene, var2csa, matched by loss of PfEMP1 surface expression and hostcell binding. Albeit the high levels of spliced, full-length transcripts, the relative abundance of intracellular translational product was sparse. In vivo, off-switching and translational repression may constitute one pathway, among others, coordinating PfEMP1 expression (Paper III). Beside PfEMP1, the parasite exports an array of other proteins to the erythrocyte surface which may partake in host-recognition and pathology. Proteomic analysis of surface proteins is, however, a challenging task. To enable the elucidation of surface-associated pRBC proteins we developed a new workflow, combining mild surface trypsinization of live-infected erythrocytes, OFFGEL fractionation of the peptide mixture followed by capillary LC-MS/MS analysis. Two highly abundant proteins (GBP130, HSP70-1) and a number of low abundant proteins were identified.