Identification, Cloning, Expression, and Characterization of the Gene for Plasmodium knowlesi Surface Protein Containing an Altered Thrombospondin Repeat Domain

Abstract

Proteins present on the surface of malaria parasites that participate in the process of invasion and adhesion to host cells are considered attractive vaccine targets. Aided by the availability of the partially completed genome sequence of the simian malaria parasite Plasmodium knowlesi, we have identified a 786-bp DNA sequence that encodes a 262-amino-acid-long protein, containing an altered version of the thrombospondin type I repeat domain (SPATR). Thrombospondin type 1 repeat domains participate in biologically diverse functions, such as cell attachment, mobility, proliferation, and extracellular protease activities. The SPATR from P. knowlesi (PkSPATR) shares 61% and 58% sequence identity with its Plasmodium falciparum and Plasmodium yoelii orthologs, respectively. By immunofluorescence analysis, we determined that PkSPATR is a multistage antigen that is expressed on the surface of P. knowlesi sporozoite and erythrocytic stage parasites. Recombinant PkSPATR produced in Escherichia coli binds to a human hepatoma cell line, HepG2, suggesting that PkSPATR is a parasite ligand that could be involved in sporozoite invasion of liver cells. Furthermore, recombinant PkSPATR reacted with pooled sera from P. knowlesi-infected rhesus monkeys, indicating that native PkSPATR is immunogenic during infection. Further efficacy evaluation studies in the P. knowlesi-rhesus monkey sporozoite challenge model will help to decide whether the SPATR molecule should be developed as a vaccine against human malarias.An effective vaccine that reduces malaria-related mortality and morbidity would indeed alleviate the suffering of millions of people around the world (17). The onset and progression of malaria infection require a complex sequence of recognition, adhesion, and invasion events between the parasite and host cells. To accomplish this, the parasite expresses a multitude of proteins on its surface that serve as ligands that interact with the receptors present on the host cells. For example, circumsporozoite protein and thrombospondin-related anonymous protein (two sporozoite-stage proteins) participate in the binding of malaria sporozoites to liver cells (6, 30). In the erythrocytic-stage cycle, erythrocyte-binding antigen 175 is recognized as a merozoite ligand that binds to glycophorin A on erythrocytes to initiate the process of merozoite invasion (33). Likewise, the products of the var genes expressed on the surface of infected erythrocytes facilitate the adherence of mature forms of parasites to deep venules of endothelial cells and thus shield them from clearance by the host immune system.The standard steps in preclinical vaccine development prior to phase I clinical trials in humans are antigen identification, its biochemical/biological characterization, and efficacy evaluation in animal models. For malaria, animal studies are generally performed using pertinent orthologs of Plasmodium falciparum (the most lethal form of human malaria) in the mouse (Plasmodium yoelii/Plasmodium berghei) and monkey (Plasmodium knowlesi) mo...

A Conserved Apicomplexan Microneme Protein Contributes to Toxoplasma gondii Invasion and Virulence

“…In P. falciparum, PfSPATR is expressed at several stages of the parasite life cycle, an important consideration for vaccine development (18). Furthering its vaccine potential, Plasmodium SPATR is immunogenic in naturally infected and immunized volunteers, and antibodies to recombinant SPATR block sporozoite invasion (18,21). These findings suggest a role in invasion, which is consistent with the ability of Plasmodium SPATR to bind HepG2 hepatoma cells (18,21).…”

“…Furthering its vaccine potential, Plasmodium SPATR is immunogenic in naturally infected and immunized volunteers, and antibodies to recombinant SPATR block sporozoite invasion (18,21). These findings suggest a role in invasion, which is consistent with the ability of Plasmodium SPATR to bind HepG2 hepatoma cells (18,21). Herein, we further dissect the significance of TgSPATR in infection by genetically ablating it in T. gondii.…”

Functional, Immunological and Three-Dimensional Analysis of Chemically Synthesised Sporozoite Peptides as Components of a Fully-Effective Antimalarial Vaccine

“…This protein binds to hepatoma cells such as HepG2; antibodies directed against it have been able to inhibit P. falciparum sporozoite invasion of liver cells [48,150]. It has been reported that PfSPATR has also bound to human RBCs and to an anopheline mosquito larvae pluripotent cell line [150], thereby highlighting its importance as ligand during host-cell recognition and invasion.…”

Cloning, expression, and immunocharacterization of surface protein containing an altered thrombospondin repeat domain (SPATR) from Plasmodium knowlesi

“…Many invasive stages of Plasmodium have been found to contain one or more thrombospondin type-1 repeat (TSR) domain [5], suggesting the possible role of this domain in sporozoite motility and hepatocyte invasion. An altered version of the thrombospondin repeat domain was first found in Plasmodium yoelii [6,7] and named the surface protein of altered thrombospondin repeat (SPATR). Orthologue of this protein has also been identified in P. knowlesi and named the pk SPATR.…”

“…SPATR protein is expressed during many stages of the P. knowlesi life cycle, including the sporozoite, merozoite and gametocyte stages [6]. Importantly, it is expressed on the cell surface during the sporozoite stage, where it is involved in liver cell invasion.…”