Abstract
Placental malaria is a significant cause of all malaria-related deaths globally for which no drugs have been developed to specifically disrupt its pathogenesis. To facilitate the discovery of antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes in the placenta microvasculature, we have developed an automated image-based assay for high-throughput screening for potent cytoadherence inhibitors in vitro. Parasitized erythrocytes were drug-treated for 24 h and then allowed to adhere on a monolayer of placental BeWo cells prior to red blood cell staining with glycophorin A antibodies. Upon image-acquisition, drug effects were quantified as the proportion of treated parasitized erythrocytes to BeWo cells compared to the binding of untreated iRBCs. We confirmed the reliability of this new assay by comparing the binding ratios of CSA- and CD36-panned parasites on the placental BeWo cells, and by quantifying the effects of chondroitin sulfate A, brefeldin A, and artemisinin on the binding. By simultaneously examining the drug effects on parasite viability, we could discriminate between cytoadherence-specific inhibitors and other schizonticidal compounds. Taken together, our data establish that the developed assay is highly suitable for drug studies targeting placental malaria, and will facilitate the discovery and rapid development of new therapies against malaria.
Highlights
Plasmodium falciparum is responsible for the most severe forms of human malaria that include cerebral malaria, pregnancy-associated malaria, and acute anemia.A major aspect of the virulence of P. falciparum derives from the ability of parasitized erythrocytes to adhere to different endothelial cell types in the deep vasculature of the body, resulting in a sequestration of the parasites away from splenic clearance [1,2,3]
Whereas parasite sequestration in the peripheral microvasculature is associated with parasitized erythrocytes that bind to CD36, ICAM-1, VCAM or E-selectin receptors, sequestration in the placenta mainly involves chondroitin sulfate A (CSA) that is abundantly expressed by placental syncytiotrophoblasts [1,6,7]
To facilitate the discovery of new antimalarial drugs targeting the cytoadherence process of Plasmodium-infected erythrocytes during pregnancy, we have developed an image-based drug susceptibility assay suitable for high-throughput screening of diverse chemical libraries
Summary
Plasmodium falciparum is responsible for the most severe forms of human malaria that include cerebral malaria, pregnancy-associated (placental) malaria, and acute anemia.A major aspect of the virulence of P. falciparum derives from the ability of parasitized erythrocytes to adhere to different endothelial cell types in the deep vasculature of the body, resulting in a sequestration of the parasites away from splenic clearance [1,2,3]. Evidence from targeted gene disruption studies have established that the PfEMP1 variant var2CSA is the main ligand mediating the cytoadherence process against placental CSA receptors [8,9,10,11] In support of these observations, var2CSA-dependent binding of parasitized erythrocytes to placental BeWo cells can be efficiently inhibited by pretreatment with soluble CSA proteins [11]. Post-mortem studies of severe malaria in previously treated patients often show high levels of infected erythrocytes bound to the microvasculature in spite of clearance of the peripheral parasitaemia [12,13] These observations strongly underscore the urgent need for new antimalarial drugs targeting the cytoadherence process of malaria parasites, in high-risk pregnancy cases
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