Yeast two - hybrid analysis of host - parasite protein interactions in Falciparum malaria

Wesley D Penn, Purdue University

Abstract

Despite renewed eradication, containment, and control efforts malaria continues to be a major cause of death and disease in the developing world. The most lethal human form of the disease is caused by the Plasmodium falciparum parasite which exhibits a complex life cycle whose asexual or intraerythrocytic stage drives the morbidity and mortality associated with malarial infection. While erythrocytic residency helps the parasite avoid immune detection it also presents the parasite with an environment hostile to its continued development. To combat these challenges the parasite radically alters its host red blood cell (RBC), inducing changes in deformability, cytoadherance, and nutrient uptake. These changes are thought to be driven by the 300–400 proteins the parasite is known or predicted to export. However, information about function and interacting partners is available for only a handful of these proteins and thus our understanding of how host-parasite protein-protein interactions affect the development and progression of malaria is woefully incomplete. To allay this problem I have undertaken a series of yeast two-hybrid (Y2H) screens to identify potential host-parasite protein interactions and thereby generate an interaction map that provides insight into the possible function of several parasite exported proteins. The thesis presented herein will describe the theory, methods, and results of these experiments as well as their possible implications for the malaria infected RBC. A series of three Y2H assays of erythrocyte membrane cytoskleton proteins versus P. falciparum activation domain libraries were undertaken. These screens yielded 179 unique interactions between 133 parasite proteins and 13 RBC proteins. Thirty-one of these interactions, representing parasite proteins known or predicted to be exported or parasite proteins with known erythrocyte surface localization were selected for confirmation. Though only 9 (29%) of these interactions retested, the interaction map provided by these linkages does provide some insight. For example, all of the interactions that retested occurred with erythrocyte proteins belonging to the membrane cytoskeleton ankyrin or junctional complexes. Ankyrin appears to play a central role in host-parasite protein interactions, accounting for five individual interactions. These interactions and several published reports imply that GBP 130 (the glycophorin binding protein) plays a role in merozoite escape, SURFIN 1.3 is likely involved in the cytoadhereance of infected RBCs (iRBCs) to endothelial cells, and PFI0130c links an undefined surface exposed parasite protein to the erythrocyte membrane cytoskeleton. MESA, a previously described erythrocyte protein band 4.1 interactor was shown to bind ankyrin and PF11_0037 appears to play a role in cytoadherance similar to that of PfEMP1. Finally PFD0090c may be a scaffolding protein that binds both ankyrin and band 4.1.

Degree

M.S.

Advisors

LaCount, Purdue University.

Subject Area

Molecular biology|Parasitology

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