Abstract
Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world's major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.
Highlights
Parasites of the Plasmodium genus are the etiological agents responsible for malaria, an infectious disease mostly occurring in developing countries with up to 40% of the world’s population described as being at risk of the disease
A vaccine targeting the proteins expressed on the surface of the blood stage of the parasite is conceptually attractive because merozoites are repeatedly and directly exposed to the human humoral immune system and naturally acquired antibodies against these proteins have been shown to confer at least partial immunity [5,6,7,8]
Design and Synthesis of P. falciparum Merozoite Cell Surface and Secreted Expression Plasmids—The regions corresponding to the entire extracellular domains of 51 merozoite cell-surface proteins from the P. falciparum 3D7 strain were determined by using transmembrane and GPI-anchor [33, 34], or signal peptide [35] prediction software
Summary
A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins*. A generic method that would overcome these technical challenges to express, in a systematic way, panels of recombinant Plasmodium proteins that have retained their native function and conformation would be a valuable resource for the molecular investigations of erythrocyte invasion and the development of a blood stage vaccine. To generate a resource of correctly folded recombinant merozoite proteins, we used a mammalian expression system and established the parameters necessary for high-level expression Using this method, we compiled a panel of 42 proteins that corresponds to the repertoire of abundant cell surface and secreted merozoite proteins of the 3D7 strain of Plasmodium falciparum. We compiled a panel of 42 proteins that corresponds to the repertoire of abundant cell surface and secreted merozoite proteins of the 3D7 strain of Plasmodium falciparum Biochemical activity of these proteins was demonstrated by recapitulating known protein interactions and by showing conformation-sensitive immunoreactivity of the recombinant proteins using immune sera
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