Two Distinct Domains of MSP1 Recognize the Band 3-GPA Complex Mediating Malaria Invasion.
Abstract: Each year there are approximately 200 million cases of human malaria and over 700,000 deaths. In order to prevent the spread of this lethal infectious disease we must design specific, effective therapies at its subcellular targets. This thesis investigates two essential malaria proteins and the roles they play in Plasmodium falciparum development and invasion in the hopes they may be use... read mored as components of a multi-drug therapy or multi-antigenic vaccine. First, Plasmodium falciparum signal peptide peptidase (PfSPP) functions at the blood stage of malaria infection where it plays an essential role. Previous work demonstrated that SPP inhibitors are lethal to malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of Plasmodium falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides a new target for the development of a multi-drug therapy at the erythrocyte stage of the parasite life cycle. The second aspect of this study focused on the specific role of merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria. MSP1 is essential for RBC invasion and considered a leading candidate for inclusion in a multi-subunit vaccine against malaria. Here, by employing phage display technology, we report a novel interaction between the N-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 previously shown to potently inhibit P. falciparum invasion of human RBCs. Additionally, a genetically modified mouse model lacking the band 3-GPA complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of red blood cells. Furthermore, we developed specific antibodies against the 5C and 6A extracellular domains of band 3 to inhibit parasite invasion by targeting host receptors. We subsequently identified RhopH3 as a binding partner to MSP1 and band 3. The combination of these findings reveals the function of two essential protein complexes in intra-erythrocyte development and invasion of the malaria parasite.
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Athar Chishti.
Committee: Michael Forgac, Peter Juo, Mercio Perrin, and Matthias Marti.
Keywords: Physiology, Molecular biology, and Parasitology.read less