cDNA sequence encoding a Plasmodium falciparum protein associated with knobs and localization of the protein to electron-dense regions in membranes of infected erythrocytes.

Plasmodium falciparum modifies the host erythrocyte's plasma membrane by the formation of electron-dense structures called knobs. We have produced monoclonal antibodies (McAbs) which specifically bind to the knobs in immunoelectron microscopic experiments with thin sections of parasitized erythrocytes. However, the McAbs fail to bind to the surface of live parasitized erythrocytes. Immunoblotting experiments with these McAbs show the antigen is localized to the erythrocyte plasma membrane. The antigen with which the McAbs react varies in mol. wt from 80 to 95 kd in different knob-producing isolates of P. falciparum and is absent in knobless variants. The McAbs react with the expressed product of a P. falciparum cDNA clone, thus demonstrating that the clone encodes part of this knob-associated protein. The sequence of the cDNA fragment partially overlaps a published cDNA sequence reported to encode the amino-terminal portion of the knob protein, and extends the predicted open reading frame by 190 amino acids. The carboxyl-terminal portion of the predicted amino acid sequence contains a highly charged stretch of approximately 100 amino acid residues. We suggest that this unusual, highly charged region participates in intermolecular salt bridging leading to dense packing of these molecules. This would create the electron-dense regions observed by electron microscopy and might also explain the insolubility of the knob-associated protein in the absence of strong ionic detergents or chaotropic agents.     

Characterization of a modified red cell membrane protein expressed on erythrocytes infected with the human malaria parasite Plasmodium falciparum: possible role as a cytoadherent mediating protein

Infections with the human malaria Plasmodium falciparum are characterized by the retention of parasitized erythrocytes in tissue capillaries and venules. Erythrocytes containing trophozoites and schizonts attach to the endothelial cells that line these vessels by means of structurally identifiable excrescences present on the surface of the infected cell. Such excrescences, commonly called knobs, are visible by means of scanning or transmission electron microscopy. The biochemical mechanisms responsible for erythrocyte adherence to the endothelial cell are still undefined. In an attempt to identify the cytoadhesive molecule on the surface of the infected cell, we have prepared monoclonal antibodies to knob-bearing erythrocytes infected with the FCR-3 strain of P. falciparum. One of these monoclonal antibodies, designed 4A3, is an IgM that reacts (by means of immunofluorescence) with the surface of unfixed erythrocytes bearing mature parasites of the knobby line; it does not react with knobless lines or uninfected erythrocytes. By immunoelectron microscopy the monoclonal antibody 4A3 was localized to the knob region. In an in vitro cytoadherence assay, the monoclonal antibody partially blocked the binding of knob-bearing cells (FCR-3 strain) to formalin-fixed amelanotic melanoma cells. The monoclonal antibody was used to immunoprecipitate a protein from extracts of knobby erythrocytes that had been previously surface iodinated. By a two-dimensional peptide mapping technique, the antigen recognized by the monoclonal antibody was found to be structurally related to band 3 protein, the human erythrocyte anion transporter.     

Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane

We attached paraformaldehyde-fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5-6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/microns 2 of membrane. In contrast, we found 3-4 filaments at each intersection and approximately 400 intersections/microns 2 of membrane. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. Our results suggest that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material. Extracting sheared ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by approximately 3 nm.     

Plasmodium falciparum merozoites: isolation by density gradient centrifugation using Percoll and antigenic analysis

Merozoites of Plasmodium falciparum were isolated and immunocytochemically analyzed. Mature parasites from knobby (K+) and knobless (K-) strains were incubated for 4 to 5 hr in RPMI 1640 with 10% serum and 10% RBC extract. About 12 to 14% of the merozoites released were recovered by density gradient centrifugation using Percoll. From 1 to 3 X 10(9) merozoites were obtained per collection. The merozoite preparations were contaminated with 10% residual bodies, about 0.1% infected and uninfected erythrocytes, about 0.1% RBC-free trophozoites and schizonts, and numerous small (less than 0.5 microns) membrane vesicles. Merozoites from the K+ and K- strains were morphologically and, by an indirect, ferritin-labeled antibody assay using serum from immune Aotus, antigenically indistinguishable. Although the residual body coats reacted with the immune Aotus serum, the membrane vesicles, some of which were seen to be blebbing from merozoites, did not react with this serum or a serum against erythrocytes. This paper describes a procedure that can be used to obtain large numbers of merozoites with little contamination by host erythrocytes.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

The relationship to knobs of the 92,000 D protein specific for knobby strains of Plasmodium falciparum

A 92,000 D protein was identified associated with the membrane of host erythrocytes infected with the FCB1 Plasmodium falciparum strain from Colombia. The same protein was identified in the knob-forming Gambian (and the Malayan Camp) strain, but was not present in all the corresponding knobless strains. In the FCB1 strain as well as in the FCR3 strain the protein is synthesized during the ring-stage period. The cleavage products of the 92,000 D protein were investigated by peptide mapping following limited proteolytic digestion with Staphylococcus aureus V8 protease. The 92,000 D protein cleavage products from both the Colombian and the Gambian strains were identical. Moreover, both the proteins were sensitive to trypsin and chymotrypsin and also to treatment with neuraminidase. Enzymatic removal of the protein from the erythrocyte membrane by trypsin or chymotrypsin did not affect parasite maturation. The merozoites thus produced were fully invasive and the morphology of the knobs was unaltered. When the erythrocyte membrane was treated with trypsin before the time of synthesis of the 92,000 D protein, it was not possible to identify the protein in membranes of later stages of infected erythrocytes, indicating that the protein cannot be inserted into the membrane cytoskeleton compartment. Knobs, however, were formed more or less normally, suggesting that it is not the accumulation of this protein which products the knobs.     

Parasitism of Pieris rapae (Lepidoptera: Pieridae) by a pupal endoparasitoid, Pteromalus puparum (Hymenoptera: Pteromalidae): effects of parasitization and venom on host hemocytes

In contrast to the situation with egg-larval and larval endoparasitic wasps, little is known about the effects of pupal endoparasitoids and their secretions on the hemocytes of their insect hosts. This study focuses on the pupal endoparasitoid, Pteromalus puparum, and its host, the small white butterfly, Pieris rapae. Parasitism by P. puparum, resulted in a significant increase in the total number of host hemocytes up to day five after parasitization. From day one to day four after parasitization, the percentage of plasmatocytes significantly decreased, and the proportion of granular cells increased. Moreover, from 12 h to day three after parasitization, hemocyte mortality in parasitized pupae was noticeably higher. When P. rapae pupae were parasitized by adult females of P. puparum irradiated by gamma-ray (pseudoparasitization), it was clear that the treated wasps could induce similar hemocyte changes. However, such phenomena did not occur in punctured host pupae (mimic-parasitization). After treatment with P. puparum venom, both the percentages of spreading plasmatocytes and encapsulated Sephadex G-25 beads were lessened significantly in vitro. Electron microscopy analysis and visualization of hemocyte F-actin with phalloidin-FITC showed that hemocytes treated with venom had a rounded configuration and neither spread nor extended pseudopods, while there was no marked alteration of hemocyte cytoskeletons after venom treatment. The results suggested that venom of P. puparum could actively suppress the hemocyte immune response of its host, but not by destroying the host hemocyte cytoskeleton.     

Fine structure of human malaria in vitro.

The erythrocytic cycle of the human malaria parasite, Plasmodium, falciparum, was examined by electron microscopy. Three strains of parasites maintained in continuous culture in human erythrocytes were compared with in vivo infections in Aotus monkeys. The ultrastructure of P. falciparum is not altered by continuous cultivation in vitro. Mitochondria contain DNA-like filaments and some cristae at all stages of the erythrocytic life cycle. The Golgi apparatus is prominent at the schizont stage and may be involved in the formation of rhoptries. In culture, knob-like protrusions first appear on the surface of trophozoite-infected erythrocytes. The time of appearance of knobs on cells in vitro correlates with the life cycle stage of parasites which are sequestered from the peripheral circulation in vivo. Knob material of older parasites coalesces and forms extensions from the erythrocyte surface. Some of this material is sloughed from the host cell surface. The parasitophorous vacuole membrane breaks down in erythrocytes containing mature merozoites both in vitro and in vivo. Merozoite structure is similar to that of P. knowlesi. The immature gametocytes in culture have no knobs.     

DNase-I-dependent dissociation of erythrocyte cytoskeletons

The human erythrocyte contains a complex of peripheral membrane proteins which forms an extensive network or cytoskeleton on the cytoplasmic membrane surface. When I treat erythrocyte cytoskeletons with deoxyribonuclease I (DNase I), the cytoskeletons dissociate and erythrocyte actin is solubilized. The dissociation of the cytoskeletons by DNase I parallels the disruption of actin filaments in vitro by DNase I and is blocked by the addition of action to the DNase I. Large protein complexes remain after DNase I disrupts the cytoskeletons, but these complexes are no longer visible in the light microscope nor sedimentable and are selectively depleted with respect to actin. From these studies, I suggest that DNase I binds to and solubilizes actin, which serves as a structural link between protein complexes in the erythrocyte cytoskeleton.     

Trafficking determinants for PfEMP3 export and assembly under the Plasmodium falciparum-infected red blood cell membrane

During the maturation of intracellular asexual stages of Plasmodium falciparum parasite-encoded proteins are exported into the erythrocyte cytosol. A number of these parasite proteins attach to the host cell cytoskeleton and facilitate transformation of a disk-shaped erythrocyte into a rounded and more rigid infected erythrocyte able to cytoadhere to the vasculature. Knob formation on the surface of infected erythrocytes is critical for this cytoadherence to the host endothelium. P. falciparum proteins have been identified that localize to the parasite-infected erythrocyte membrane: the variant cytoadherence ligand erythrocyte membrane protein 1 (PfEMP1), the knob-associated histidine-rich protein (KAHRP) and the erythrocyte membrane protein 3 (PfEMP3). In this study, we have generated parasites expressing PfEMP3-green fluorescent protein chimeras and identified domains involved in entry to the secretory pathway, export across the parasitophorous vacuolar membrane and attachment to Maurer's clefts and the erythrocyte membrane. Solubility assays, fluorescence photobleaching experiments and immunogold electron microscopy suggest that the exported chimeric proteins are trafficked in a complex rather than in vesicles. This study characterizes elements involved in the tight but transient binding of PfEMP3 to Maurer's clefts and shows that the same elements are necessary for correct assembly under the erythrocyte membrane.     

Host physiological changes due to parasitism of a braconid wasp, Cotesia plutellae, on diamondback moth, Plutella xylostella

Braconid wasps, Cotesia plutellae (Kurdjumov), were collected from parasitized host larvae of diamondback moth, Plutella xylostella (L.) in Korea. Virus particles were found in the oviduct lumen of C. plutellae females. Multiple nucleocapsids with approximately 30-nm diameter and variable length (30-80 nm) were surrounded with a single unit membrane envelope. The parasitization of C. plutellae completely inhibited pupal metamorphosis. The parasitized larvae showed significant decrease in feeding activity and total hemolymph proteins, especially as larval storage proteins. They also showed a significant decrease in immune capacity as evidenced by reduced ability to form hemocyte nodules and reduced phenoloxidase and lysozyme activity. Here, we show that C. plutellae has an endosymbiotic virus like other reported species in Microgastrinae, and suggest that it causes host developmental arrest and immune-depression at parasitization.     

Morphological changes in erythrocytes induced by malarial parasites

Host cell alterations induced by Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoelectron microscopy. P. falciparum infection induces knobs, electron-dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasilianum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasilianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erythrocyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythrocyte membrane. P. malariae induces caveolae, electron-dense material, vesicles, clefts and knobs in the erythrocyte. Although vesicles and caveolae are seen in the erythrocyte cytoplasm, they do not form caveola-vesicle complexes as seen in P. vivax-infected erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host cell changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not clear.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

Structural and functional studies of interaction between Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and erythrocyte spectrin

Plasmodium falciparum dramatically modifies the structure and function of the membrane of the parasitized host erythrocyte. Altered membrane properties are the consequence of the interaction of a group of exported malaria proteins with host cell membrane proteins. KAHRP (the knob-associated histidine-rich protein), a member of this group, has been shown to interact with erythrocyte membrane skeletal protein spectrin. However, the molecular basis for this interaction has yet to be defined. In the present study, we defined the binding motifs in both KAHRP and spectrin and identified a functional role for this interaction. We showed that spectrin bound to a 72-amino-acid KAHRP fragment (residues 370-441). Among nine-spectrin fragments, which encompass the entire alpha and beta spectrin molecules (four alpha spectrin and five beta spectrin fragments), KAHRP bound only to one, the alpha N-5 fragment. The KAHRP-binding site within the alpha N-5 fragment was localized uniquely to repeat 4. The interaction of full-length spectrin dimer to KAHRP was inhibited by repeat 4 of alpha spectrin. Importantly, resealing of this repeat peptide into erythrocytes mislocalized KAHRP in the parasitized cells. We concluded that the interaction of KAHRP with spectrin is critical for appropriate membrane localization of KAHRP in parasitized erythrocytes. As the presence of KAHRP at the erythrocyte membrane is necessary for cytoadherence in vivo, our findings have implications for the development of new therapies for mitigating the severity of malaria infection.     

Synaptosomal cytoskeleton visualized by whole mount electron microscopy

Organization of synaptosomal cytoskeleton was reproducibly visualized by the technique of whole mount electron microscopy. Synaptosomes from rat cerebrums were immobilized on the formvar membrane of the electron microscopic grid, partly solubilized by detergents of various kinds, and treated with chemicals to reveal cytoskeletons and their characteristics. Synaptosomal cytoskeletons consisted of three types: (1) pre-synaptic fiber network structure whose composite fiber was 15–20 nm in diameter and formed 60–100 nm circular rings. The rings had small particles inside and were organized into three-dimensional networks. The pre-synaptic network was different from the Triton-unextractable structure of mitochondria. (2) Post-synaptic fiber aggregate was constructed of 10-nm filaments that were typically visualized as deoxycholate- or N-lauroyl sarcosinate-unextractable cytoskeletons. The aggregate was a major structure in the Triton-unextractable cytoskeleton of synaptic plasma membrane (more than 95%). (3) Fiber connecting individual clusters of synaptosomal cytoskeletons which was probably an artifactual product formed during and after synaptosomal isolation. Existence of actin was indicated both in pre- and post-synaptic cytoplasm.     

Evidence for trafficking of PfEMP1 to the surface of P. falciparum-infected erythrocytes via a complex membrane network

The human malarial parasite Plasmodium falciparum exports virulence determinants, such as the P. falciparum erythrocyte membrane protein 1 (PfEMP1), beyond its own periplasmatic boundaries to the surface of its host erythrocyte. This is remarkable given that erythrocytes lack a secretory pathway. Here we present evidence for a continuous membrane network of parasite origin in the erythrocyte cytoplasm. Co-localizations with antibodies against PfEMP1, PfExp-1, Pf332 and PfSbpl at the light and electron microscopical level indicate that this membrane network is composed of structures that have been previously described as tubovesicular membrane network (TVM), Maurer's clefts and membrane whorls. This membrane network could also be visualized in vivo by vital staining of infected erythrocytes with the fluorescent dye LysoSensor Green DND-153. At sites where the membrane network abuts the erythrocyte plasma membrane we observed small vesicles of 15-25 nm in size, which seem to bud from and/or fuse with the membrane network and the erythrocyte plasma membrane, respectively. On the basis of our data we hypothesize that this membrane network of parasite origin represents a novel secretory organelle that is involved in the trafficking of PfEMP1 across the erythrocyte cytoplasm.     

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BACKGROUND: The development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised. METHODS: P. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database. RESULTS: Following four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library. CONCLUSION: The construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.     

Plasmodium falciparum malaria: association of knobs on the surface of infected erythrocytes with a histidine-rich protein and the erythrocyte skeleton

Plasmodium falciparum-infected erythrocytes (RBC) develop surface protrusions (knobs) which consist of electron-dense submembrane cups and the overlying RBC plasma membrane. Knobs mediate cytoadherence to endothelial cells. Falciparum variants exist that lack knobs. Using knobby (K+) and knobless (K-) variants of two strains of P. falciparum, we confirmed Kilejian's original observation that a histidine-rich protein occurred in K+ parasites but not K- variants (Kilejian, A., 1979, Proc. Natl. Acad. Sci. USA, 76:4650-4653; and Kilejian, A., 1980, J. Exp. Med., 151:1534-1538). Two additional histidine-rich proteins of lower molecular weight were synthesized by K+ and K- variants of both strains. We used differential detergent extraction and thin-section electron microscopy to investigate the subcellular location of the histidine-rich protein unique to K+ parasites. Triton X-100, Zwittergent 314, cholic acid, CHAPS, and Triton X-100/0.6 M KCl failed to extract the unique histidine-rich protein. The residues insoluble in these detergents contained the unique histidine-rich protein and electron-dense cups. The protein was extracted by 1% SDS and by 1% Triton X-100/9 M urea. The electron-dense cups were missing from the insoluble residues of these detergents. The electron-dense cups and the unique histidine-rich protein appeared to be associated with the RBC skeleton, particularly RBC protein bands 1, 2, 4.1, and 5. We propose that the unique histidine-rich protein binds to the RBC skeleton to form the electron-dense cup. The electron-dense cup produces knobs by forming focal protrusions of the RBC membrane. These protrusions are the specific points of attachment between infected RBC and endothelium.     

Correlation between concentration of hemolymph nutrients and amount of fat body consumed in lightly and heavily parasitized hosts (Pseudaletia separata)

Two states of parasitization in the Pseudaletia separata-Cotesia kariyai system were examined: one that was lightly parasitized and one that was heavily parasitized. We predicted that the consumption of fat body and hemolymph nutrients depends on the number of parasitoid larvae in the host. Lightly parasitized hosts (average clutch size+/-S.E.: 42.5+/-16.2, N=15) and heavily parasitized hosts (average clutch size+/-S.E.: 230.2+/-8.8, N=15) were prepared artificially. Eight days after parasitization, perivisceral fat body was depleted in the heavily parasitized host, although peripheral fat body was not yet consumed, but by day 10 most of the peripheral fat body was consumed. In lightly parasitized hosts, perivisceral fat body was not consumed by day 10. The parasitoid larvae deplete the perivisceral fat body first and then consume the peripheral fat body in the heavily parasitized host. The amount of trehalose, the major carbohydrate in the hemolymph, was related to the number of parasitoid larvae developing in the host. In a heavily parasitized host, trehalose concentrations remained low. However, in lightly parasitized hosts, the amount of trehalose increased 8 days after parasitization and then decreased by day 10. Protein and total lipid concentrations in the hemolymph of the heavily parasitized host were significantly lower than in lightly parasitized host on day 10, suggesting that the large number of parasitoid larvae depleted the fat body and hemolymph nutrients by day 10. High concentrations of total lipid on day 8 and 10 in lightly parasitized hosts and on day 8 in heavily parasitized host are likely to be attributed to the teratocytes.