Plasmodium falciparum: Analysis of Protein-Protein Interactions of the 140/130/110-kDa Rhoptry Protein Complex Using Antibody and Mouse Erythrocyte Binding Assays

The high-molecular-weight rhoptry proteins of Plasmodium falciparum exist in a multiprotein complex consisting of proteins of 140, 130, and 110 kDa. The complex of rhoptry proteins binds to human and mouse erythrocyte membranes in association with a 120-kDa SERA protein. These proteins are believed to participate in the process of erythrocyte invasion. We have used six different antibodies (polyclonal and monoclonal) known to precipitate the high-molecular-weight rhoptry protein complex (HMWC) to analyze the structural relationship of proteins within the complex. Limited proteolysis of immune complexes (IC) immobilized on Sepharose beads (protein \u27footprinting\u27) and binding of SV8 protease generated peptides to intact mouse erythrocytes was performed. The 140-kDa polypeptide was more susceptible to protease digestion followed by the 130- and 110-kDa polypeptides. The susceptibility of the 140-kDa polypeptide to protease digestion was independent of the type of precipitating antibody. We identified a 120-kDa protein as the major proteolytic fragment of the 140-kDa protein. SV8 protease generated peptide fragments derived from the 110- and 130-kDa proteins contained putative mouse erythrocyte binding domains. Immunoprecipitation of SV8-generated peptides gave peptide profiles similar to those obtained with protein \u27footprinting\u27. Additional experiments performed to investigate the stability of the HMWC using chaotropic and lyotropic agents demonstrated that the HMWC was stable to perturbatory reagents known to disaggregate macromolecular complexes. Solubilization of schizonts with 6 M urea and 4 M MgCl followed by IC formation led to differential precipitation of the 110-kDa polypeptide, while solubilization with 3 M KCl resulted in the differential precipitation of the 140- and 130-kDa polypeptides, suggesting that both proteins may be in direct association. Treatment of immobilized IC with different perturbatory agents including 6 M urea, 3 M KCl, 4 M MgCl , or 2% SDS from an insoluble matrix resulted in the elution of the intact complex. The mouse erythrocyte binding property of the HMWC is conserved among different geographical isolates of P. falciparum. The results provide insights concerning the mechanism of protein-protein interaction within the complex. © 1993 Academic Press, Inc. 2

The Role of Host Inflammatory Mediators As Biomarkers for Malaria Pathogenesis

Malaria pathogenesis results from a complex interplay of pro-inflammatory and antiinflammatory mediators produced as a result of innate responses following Plasmodium infection. These mediators interact with host tissues and organs resulting in damage and destruction that characterizes specific syndromes associated with red blood cell destruction, vascular endothelial disturbances, microvascular obstruction, respiratory distress, retinopathy, inflammatory responses to toxins and other parasite metabolites and placental tissue destruction. The malaria syndromes define uncomplicated malaria (UM), cerebral malaria (CM), placental malaria (PM), severe malarial anemia (SA), and severe malaria (SM). Approximately 515 million cases of malaria are reported annually with 1-3 million deaths occurring in children. Five Plasmodium species infect humans: P. falciparum, P. vivax P. malariae P. ovale and P. knowlesi. Plasmodium falciparum is the agent most associated with severe and fatal malaria. Pregnant women and children make up the most vulnerable groups to develop severe pathogenesis from malaria infection. Cytokines, chemokines, vascular endothelial proteins, angiogenic factors, and other host mediators make up important mediators of pathogenesis in the host. In addition, other non-immunologic host molecules, parasite proteins, metabolites and toxins may contribute to severe pathogenesis in the host. Determining which of the mediators enhance adverse outcomes for disease progression and poor disease prognosis as well as identifying those mediators with protective effects is an area of intense investigation in malaria research. The cytokines tumor necrosis factor alpha (TNFα), interleukin (IL)-12, IL-1ß, IL-10 and interferon gamma (IFNγ) are associated with the pathogenesis of cerebral malaria and severe anemia, with TNFα being strongly implicated with this form of malaria. Treatment of patients with neutralizing anti-TNFα antibodies was effective in resolving clinical symptoms. Low ratios of IL-10 to TNFα in serum correlate with severe anemia in contrast to high IL-10 to TNFα ratios seen in children with uncomplicated malaria. Levels of soluble ICAM-1, IL-1Ra, IL-2R, IL-4, IL-6, IL-6R, neopterin, angiopoeitin (ANG) 2 and TNFα are found to be highly elevated in patients with cerebral malaria, with TNFα being highly elevated in patients who died from cerebral malaria compared to patients with uncomplicated malaria. TNFα and ANG2 levels can predict poor prognosis with fatal outcomes in cases of cerebral malaria. A diagnostic test that is prognostic and capable of discriminating among the different malaria syndromes as well as distinguishing infections caused by non malaria pathogens will improve disease management of malaria and disease surveillance. A diagnostic test that incorporates host mediators of disease as well as parasite products and toxins would detect biomarkers of disease progression, diseases severity and development of cerebral malaria and will also be useful in determining the effectiveness of parasite clearance following drug treatment. In this chapter we examine studies performed to evaluate host mediators associated with malaria pathogenesis; uncomplicated malaria (UM), cerebral malaria (CM), placental malaria (PM), severe malarial anemia (SA), and severe malaria (SM) to determine the effectiveness of the mediators as biomarkers for malaria diagnosis. © 2012 Nova Science Publishers, Inc. All rights reserved

In Vitro Human Cell-Free Expression System for Synthesis of Malaria Proteins

In this study, we performed cell-free expression of Plasmodium proteins using the in vitro human cell-free protein expression systems for DNA and mRNA. Malaria rhoptry genes (PFc14-0344, PFc0120w, PY01759, PY00763, PY07482, and PY04666) and a Maurer\u27s cleft gene (PfA0680c) identified from proteome analysis studies were cloned into the pT7CFE1-CHis expression vector. Following a coupled transcription-translation procedure, expressed proteins were analyzed by His-tag staining and by western blotting using protein specific antibodies and nickel-horseradish peroxidase (HRP) for histidine detection. Antibodies against whole rhoptries of Plasmodium falciparum and Plasmodium yoelii merozoites and antibodies specific for the PfMC-2TM protein identified translated proteins. The rhoptry specific antibodies exhibited cross reactivity among the expressed proteins of P. falciparum and P. yoelii. The results demonstrate that the in vitro human cell-free protein expression system is suitable for rapid expression and screening of malaria vaccine candidates and diagnostic biomarkers. © Springer-Verlag 2012

A Plasmodium falciparum Protein Located in Maurer’s Clefts Underneath Knobs and Protein Localization in Association With Rhop-3 and SERA in the Intracellular Network of Infected Erythrocytes

We report on the characterization of monoclonal antibodies against Plasmodium falciparum schizonts, which recognize parasite proteins of 130 kDa and 20 kDa. The 130-kDa protein was released by alkaline sodium carbonate treatment, suggesting that the protein is a peripheral membrane protein, while the 20-kDa protein remained associated with the membranes following alkali treatment, suggesting it may be an integral membrane protein. Both proteins were localized to large cytoplasmic vesicles within the cytoplasm of trophozoite and schizont-infected erythrocytes by immunofluorescence assay and confocal microscopy. Both proteins colocalized with Bodipy-ceramide in trophozoite and immature schizont-infected erythrocytes, but not in segmenters. The 130-kDa protein was localized by immunoelectron microscopy (IEM) to Maurer\u27s clefts underneath knobs in a knobby and cytoadherent (K /C ) P. falciparum strain. No IEM reactivity was obtained in a knobless and non-cytoadherent (K /C ) parasite strain. We investigated stage-specific protein expression and protein localization by indirect immunofluorescence assay. Bodipy-ceramide colocalization assays with Rhop-3 and serine-rich antigen (SERA)-specific antibodies were performed. A similar colocalization in trophozoites and schizonts was obtained using the rhoptry-specific antibody 1B9 reactive with the 110-kDa Rhop-3 protein. In segmenters, unlike trophozoites and immature schizonts, there was no Bodipy-ceramide colocalization with antibody 1B9. A difference in protein colocalization was seen using specific antibody 152.3F7.1.1, reactive with SERA. Antibodies to SERA colocalized with Bodipy-ceramide in schizonts, including segmenters. Collectively the data suggest that Rhop-3 transits through the intracellular network en route to the rhoptries and both vesicle-specific proteins may function in the intracellular network. + + -

Seroprevalence and specificity of human responses to the Plasmodium falciparum rhoptry protein Rhop-3 determined by using a C-terminal recombinant protein.

Rhoptry proteins participate in invasion of erythrocytes by malaria parasites. Antibodies to some of these proteins can inhibit invasion and partially protect monkeys from disease. To examine human serological responses to the 110-kDa component (Rhop-3) of the high-molecular-weight rhoptry protein complex, two cDNA clones corresponding to Rhop-3 were identified by immunologic screening. A recombinant protein representing the C-terminal one-third of the Rhop-3 was used to assess the seroprevalence to this protein in geographically isolated populations with different patterns of malaria transmission. The immunoglobulin G (IgG) positivity rate for the recombinant Rhop-3 in an enzyme-linked immunosorbent assay was 30% in an area of Papua New Guinea where malaria is holoendemic. In Kenya, the prevalence rates were 43 and 36%, respectively, in an area of hyperendemicity and an area of seasonal transmission. By contrast, rates of IgG seroprevalence to an extract of Gambian strain of Plasmodium falciparum were 48, 90, and 97% respectively, in these populations. In these areas, the pattern of antibody recognition of Rhop-3 is more similar (1.7-fold maximum difference) than the parasite extract (5-fold difference). The difference in seroresponses may represent antigenic polymorphism in different parasite strains, while their similarity for the Rhop-3 fragment may represent conservation of this protein. Recombinant- and parasite extract-specific IgG was not found in individuals infected only with Plasmodium vivax. Cross-reactivity was seen in the IgM assay. In Mombasa (Kenya), maternal and cord Rhop-3-specific IgG activities were similar. Fetal antigen-specific IgM reactivity was generally undetectable for all antigens