Plasmodium falciparum contributes to 1.5-2.7 million deaths annually. Cerebral malaria, a complication of P. falciparum infections, is associated with sequestration of erythrocytes in the blood vessels of the brain and is due to the adhesion of malaria-infected erythrocytes (PE) to the endothelial cells (EC) lining the microcirculation. Sequestration of PE in the placenta of pregnant women leads to complications that include low fetal birth weight and an increased risk of death. At present none of the therapies for malaria specifically target the adhesive phenomena associated with sequestration. Describing the molecular architecture of the PE and EC surfaces is critical to understanding the mechanisms that lead to sequestration and for developing anti-adhesive treatments. It is known that increased adhesiveness of the PE is due to parasite-induced changes in the erythrocyte membrane protein, band 3, as well as the surface exposure of parasite-encoded proteins. Though the amino acid sequences of band 3 critical to adhesion have been identified it is not known how this adhesin is formed. To define the possible role of band 3 aggregation and/or proteolysis in inducing conformational change, the lateral distribution of band 3 within the bilayer will be evaluated by freeze fracture microscopy and coordinated with electron micrographic immunolocalization of adhesive sites, as well as by the use of fluorescence photobleaching recovery, polarized fluorescence depletion and luminescence quenching. In addition, to biochemically characterize the structural changes in band 3 that could influence the disposition of adhesive sequences, attempts will be made to isolate truncated forms of band 3 and cleavage sites identified. Immunoprecipitation, immunoblotting as well as competitive inhibition of antibody binding will be used to identify common adhesive motifs in the band 3-related and the parasite-encoded adhesin Plasmodium falciparum erythrocyte membrane protein-1; estimates of their contribution to cytoadherence will be determined. Other PE adhesins as well as EC ligands of microvessels in the human brain, dermis and bone marrow that bind PE will be identified and characterized through the use of monoclonal antibodies, phage display and synthetic combinatorial library technologies, and peptide affinity chromatography. Once identified, it should be possible to design drugs (i.e. peptidomimetics, small molecules, antibodies) that could inhibit or reverse sequestration thus allowing for better and safer therapeutic modalities for individuals suffering from falciparum malaria.
