The long-term objective of this proposal is to continue to examine the functional consequences of interaction between red cell membrane proteins and proteins elaborated by intraerythrocytic stages of the malarial parasite Plasmodium falciparum. Profound changes occur in the properties of red cell following infection, including markedly reduced deformability and increased adhesiveness. The hypothesis we will explore is that the Parasite-mediated changes in red cell properties are the result of different parasite derived proteins interacting with specific red cell membrane proteins. To accomplish these goals, we propose the following four specific aims: 1) Define the biophysical consequences of interaction between specific malarial proteins and the red cell membrane skeleton. We will determine the effects of these protein-protein interactions on membrane extensional rigidity, membrane viscosity, membrane mechanical integrity and lateral mobility of membrane proteins. The parasite proteins to be studies include the cytoadherence molecule PfEMP-1, and other malaria proteins including HRP-I, MESA, PfEMP-3, and RESA that bind specifically to the red cell membrane skeleton; 2) Define at the molecular level the specific interacting domains of red cell membrane skeletal proteins and P. Falciparum proteins to obtain detailed understanding of how specific protein-protein interactions in infected red cells can induce functional membrane alterations; 3) Define the contribution of various malarial proteins to the strength of adhesion of obtain quantitative insights into the process of cytoadherence. Cytoadherence is a major virulence factor in P. Falciparum infection and involves the specific interaction of the PfEMP-1 protein with receptors on vascular endothelial cells. We will examine whether parasite lines that express antigenic variants of PfEMP-1 bind with differing strengths to the same ligand and also determine whether accessory proteins made by the parasite, such as MESA, HRP-1 and PfEMP-3, modulate the strength of the adhesive interaction; 4) Examine the contributions of specific red membrane proteins to malarial- parasite induced changes in red cell function by comparing the biophysical sequelae that accompany parasite infection of normal red cells with those seen following infection of mutant red cells with either qualitative or quantitative defects in various membrane skeletal proteins. The novel experimental approaches and unique reagents we have developed will be used to carry out these proposed studies. The successful accomplishment of these aims, using molecular engineering in conjunction with novel biophysical approaches, should enable us to further our understanding to the mechanism(s) involved in action of an important human pathogen.
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