Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells

During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.     

Phosphorylation of membrane proteins from Plasmodium berghei-infected red cells.

Membrane from Plasmodium berghei-infected mouse red cells has a different pattern of phosphorylation by (γ-³²P)ATP from normal membrane. A phosphorylated membrane protein of apparent molecular weight 42,000, absent in membrane from normal cells, can be detected in membrane from infected cells. The new phosphorylated protein can be extracted by 0.1 mM EDTA but not by triton X-100, indicating that it may be red cell actin.     

Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malaria-infected red blood cells

Adhesion of parasite-infected red blood cells to the vascular endothelium is a critical event in the pathogenesis of malaria caused by Plasmodium falciparum. Adherence is mediated by the variant erythrocyte membrane protein 1 (PfEMP1). Another protein, erythrocyte membrane protein-3 (PfEMP3), is deposited under the membrane of the parasite-infected erythrocyte but its function is unknown. Here we show that mutation of PfEMP3 disrupts transfer of PfEMP1 to the outside of the P.FALCIPARUM:-infected cell. Truncation of the C-terminal end of PfEMP3 by transfection prevents distribution of this large (>300 kDa) protein around the membrane but does not disrupt trafficking of the protein from the parasite to the cytoplasmic face of the erythrocyte membrane. The truncated PfEMP3 accumulates in structures that appear to be associated with the erythrocyte membrane. We show that accumulation of mutated PfEMP3 blocks the transfer of PfEMP1 onto the outside of the parasitized cell surface and suggest that these proteins traffic through an erythrocyte membrane-associated compartment that is involved in the transfer of PfEMP1 to the surface of the parasite-infected red blood cell.     

Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.     

Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes

Malaria parasites during intraerythrocytic development change the ultrastructure, biophysics, and the antigens of the host red blood cell membrane. Parasite-encoded proteins are associated with, inserted into, or secreted across the infected erythrocyte membrane. Since parasites of the genus Plasmodium are eukaryotic cells, it must be assumed that they possess essentially eukaryotic modes of vesicle-mediated transport and translocation of proteins and membranes. Numerous studies have demonstrated vesicular structures in the cytoplasm of malaria-infected red blood cells and an assortment of parasite proteins associated with the different vesicles, membranes, and membrane-defined compartments. Some parasite polypeptides remain trapped between the parasite and the parasitophorous vacuole membranes PVM, whereas others are associated with morphologically distinct membrane-limited vesicles and vacuoles. Some of these same parasite protein antigens also associate with the erythrocyte membrane or with parasite-induced ultrastructural modifications in the membrane of the parasitized red blood cells. This implies that intracellular transport occurs in malaria-infected erythrocytes, a capacity that uninfected red blood cells normally lose upon enucleation. The specific locations of parasite antigens within the infected cell also implys the existence of targeting signals in the translocated parasite polypeptides and perhaps transport-mediating proteins. The genes corresponding to some of these translocated proteins have been sequenced. Typical (and in some cases atypical) signal peptide sequences occur, as well as a number of sequences that may result in posttranslational modifications. How or if these features figure in to the translocation across, and targeting to a particular membrane compartment of the intraerythrocytic parasite remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Magnetic susceptibility of iron in malaria-infected red blood cells

During intra-erythrocytic maturation, malaria parasites catabolize up to 80% of cellular haemoglobin. Haem is liberated inside the parasite and converted to haemozoin, preventing haem iron from participating in cell-damaging reactions. Several experimental techniques exploit the relatively large paramagnetic susceptibility of malaria-infected cells as a means of sorting cells or investigating haemoglobin degradation, but the source of the dramatic increase in cellular magnetic susceptibility during parasite growth has not been unequivocally determined. Plasmodium falciparum cultures were enriched using high-gradient magnetic fractionation columns and the magnetic susceptibility of cell contents was directly measured. The forms of haem iron in the erythrocytes were quantified spectroscopically. In the 3D7 laboratory strain, the parasites converted approximately 60% of host cell haemoglobin to haemozoin and this product was the primary source of the increase in cell magnetic susceptibility. Haemozoin iron was found to have a magnetic susceptibility of (11.0 ± 0.9) × 10^? 3 mL mol^? 1. The calculated volumetric magnetic susceptibility (SI units) of the magnetically enriched cells was (1.88 ± 0.60) × 10^? 6 relative to water while that of uninfected cells was not significantly different from water. Magnetic enrichment of parasitised cells can therefore be considered dependent primarily on the magnetic susceptibility of the parasitised cells.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

Plasmodium lophurae: the ultrastructure of the exoerythrocytic stages

The fine structure of the exoerythrocytic stages of Plasmodium lophurae was studied. in specimens grown in tissue cultures of avian cells. Specimens were prepared for sectioning by a method which minimizes disturbance and permits precise selection and orientation specimens.Plasmodium lophurae is similar in many aspects to P. fallax. Merozoites are highly specialized and differentiated. Analysis of their ultrastructure revealed the polar complex to be a specialization of the pellicular envelope and its associated underlying microtubules. The polar rings may simply be a modification of the inner membrane of the pellicle and not discrete structures as previously reported. The electron-dense polar organelles are separated on morphological grounds into three groups: the large paired organelles and the small dense bodies which are both linked to microducts, and the transitional bodies, a third organelle being reported for the first time. Transitional bodies are without microducts, occur in fully mature merozoites and persist only for a short period. All three of these organelles appear to be related to and possibly even derived from internal membrane systems and ribosomes. The apolar end of the merozoite contains the mitochondrion and its associated spherical body. Detailed study of the latter shows it to be cylindrical.Upon entering the host cell, the parasite adds a third membrane at the interface between it and the cell. The merozoite becomes spherical and undergoes transformation into a trophozoite. During this reorganization phase, dedifferentiation occurs and is followed by a rapid growth phase. The end of the growth phase is signaled by the appearance of germinal clefts and nuclear division. The entire process of schizogony culminates in a highly synchronized formation of merozoites.Processes of the limiting membrane forming the host parasite interface were observed extending deply into the cytoplasm of the host cell and often appeared to form bridges between two or more parasites. The significance of this new observation is not yet established.     

Expression of red cell membrane proteins in erythroid precursor cells

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50–60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70–80% of all cells in the spleen of anemic animals, while only 1–2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.     

The serine hydroxymethyltransferase of Plasmodium lophurae.

Plasmodium lophurae serine hydroxymethyltransferase (EC 2.1.2.1) was partially purified and characterized by (NH4)2SO4 fractionation and chromatography on Sephadex G-100. The enzyme, precipitated by 3.0.3.3 M (NH4)2SO4, had a molecular weight of 68,300 as estimated by exclusion chromatography on G-100. The pH optimum of the enzyme was 6.8-7.6 in sodium phosphate-citrate buffer. Citrate stabilized the enzyme during storage in phosphate buffer at 4 C. The Km was 4.3 X 10(-3) M for L-serine and 2.5 X 10(-4) M for tetrahydrofolate.     

Pyridoxine kinase in Plasmodium lophurae and duckling erythrocytes.

Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent Km for pyridoxine of the parasite enzyme was 6.6 times 10(-5) M whereas the host red cell enzyme Km was 1.9 times 10(-6) M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent Ki of 1.5 times 10(-6) and 8.6 times 10(-6) M, respectively. These results suggest that the vitamin B6 metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.     

The role of variant surface antigens on malaria-infected red blood cells.

It has been proposed that the primary role of variant antigens appearing on the surface of red blood cells infected with malaria parasites is to mediate cytoadherence, and that the antigenic variation they display is an adaptation to avoid immune attack. Here, Allan Saul proposes that their role is the opposite: that their primary purpose is to generate an immune response, which regulates their growth and thereby establishes a chronic infection, and that the role of cytoadherence is to ensure that parasites failing to express this flag to the immune system are destroyed by the spleen.     

Dihydrofolate Reductase in Plasmodium lophurae and Duckling Erythrocytes*

Dihydrofolate reductase activity in duckling erythrocytes was found to be low, while activity in erythrocytes heavily infected with small uninucleate trophozoites was like that of uninfected erythrocytes. Activity of the enzyme in erythrocytes infected with large multinucleate parasites, however, was greatly increased. This activity was 5 times higher in erythrocyte-free large trophozoites than in small ones. The dihydrofolate reductase of P. lophurae differed from the host enzyme in: greater molecular weight; higher sensitivity to pyrimethamine inhibition; pH optimum; substrate and cofactor specificity; and stimulation by salts. The parasite enzyme was partially purified by ammonium sulfate precipitation.     

Pyridoxine Kinase in Plasmodium lophurae and Duckling Erythrocytes*

SYNOPSIS. Pyridoxine kinase enzyme activity was greatly increased in duckling erythrocytes infected with Plasmodium lophurae. Pyridoxine kinase activity in parasites freed from erythrocytes was much greater than that of uninfected erythrocytes. The apparent K_m for pyridoxine of the parasite enzyme was 6.6 × 10^-5 M whereas the host red cell enzyme K_m was 1.9 × 10^-6 M. Deoxypyridoxine inhibited host and parasite pyridoxine kinase activity with an apparent K_i of 1.5 × 10^-6 and 8.6 × 10^-6 M, respectively. These results suggest that the vitamin B₆ metabolism of the malaria parasites is distinct and separate from that of the host erythrocytes.