Purification of the erythrocytic stages ofBabesia bigemina from cultures

Exposure of erythrocytes infected withBabesia bigemina to glycerol-enhanced osmotic shock yielded preparations containing infected erythrocyte ghosts, free parasites, and some intact erythrocytes. The released parasites were purified and concentrated by centrifugation in Percoll gradient. Recovered free parasites were shown by the fluorescein acetate technique to be metabolically active, but their infectivity in vitro was low. It was demonstrated by electron microscopy that most of the released parasites had intact plasma membranes. There was only slight contamination of the free-parasite preparation with host erythrocyte debris.     

Cryptic disposition of antigenic parasite proteins in plasma membranes of erythrocytes infected with Plasmodium chabaudi

Plasma membranes of Plasmodium chabaudi-infected erythrocytes contain seven major neoproteins with apparent molecular masses of 154, 145, 90, 72, 67, 52, and 33 kDa, respectively. These neoproteins, with the exception of the two larger ones, can be metabolically labelled with [14C]isoleucine. The seven neoproteins are antigenic as revealed by Western blotting using hyperimmune sera obtained from two different mouse strains. None of the parasite proteins is accessible from the outside in intact P. chabaudi-infected erythrocytes as determined by lactoperoxidase-mediated radioiodination, indirect immune fluorescence microscopy, or post-embedding immunoelectron microscopy. These methods, however, identify parasite proteins in host cell plasma membranes when the latter are artificially changed either during isolation or by methanol fixation. We conclude therefore that parasitic proteins are cryptically arranged in intact host cell plasma membranes of P. chaubaudi-infected erythrocytes.     

Distinct lipid compositions of parasite and host cell plasma membranes from Plasmodium chabaudi-infected erythrocytes

Mouse erythrocytes infected with early or late trophozoites of the malaria parasite Plasmodium chabaudi were fractionated into free parasites and host cell plasma membranes, and both fractions were analyzed for cholesterol content and the composition of phospholipids and total fatty acids. The major results are: (i) parasites contain only a very low level of cholesterol which is about one-tenth of that of host cell plasma membranes. (ii) Parasites also contain less sphingomyelin and phosphatidylserine as well as more phosphatidylcholine than host cell plasma membranes. (iii) Parasites contain less 18:0 and 18:1 and more 18:2 and 20:4 fatty acids than host cell plasma membranes. (iv) During intraerythrocytic growth of parasites from early to late trophozoites, the relative proportions of cholesterol and phospholipids remain largely unchanged in both parasites and host cell plasma membranes. However, significant changes occur in the fatty composition of both compartments. There is an increase in the 20:4 and a decrease in the 18:0 and 18:1 fatty acids. (v) Plasma membranes of infected and non-infected erythrocytes exhibit about the same cholesterol content and phospholipid composition, but differ in the total fatty acid composition. Our data suggest the existence of distinct mechanisms controlling the different lipid compositions of parasites and host cell plasma membranes in whole Plasmodium chabaudi-infected erythrocytes during intraerythrocytic development of parasites, though both compartments are known to depend on the supply of various lipids from the host.     

Plasmodium falciparum antigens associated with membrane structures in the host erythrocyte cytoplasm

Hybridomas were made from mice immunized with plasma membranes from erythrocytes infected with Plasmodium falciparum. Among the monoclonal antibodies produced, a series reacted with antigens in the host cell cytoplasm. Immunoelectron microscopy, along with indirect fluorescent antibody double labeling experiments, were used to further localize the antigens to membrane structures (presumably Maurer's clefts) in the erythrocyte cytoplasm. The epitopes thus localized are found on three parasite proteins (20 kDa, 29 kDa, and 45 kDa) and one parasite glycoprotein (45 kDa). They are likely to be part of a transport system for the parasite.     

Surface Properties of Extracellular Malaria Parasites: Morphological and Cytochemical Study

Morphological and cytochemical surface characteristics of isolated malaria parasites (Plasmodium berghei) and host erythrocytes were compared by electron microscopy by using thin section and carbon replica techniques. Erythrocytes were uniform in shape and had fine, granular surfaces. In contrast, free parasites exhibited a variety of sizes, shapes, and surface textures. Fine surface stippling was a common topographical feature of isolated parasites. Small, infective forms often had patterned surfaces resulting from the protuberance of an underlying thick intermediate layer. Results of cytochemical analysis using a sialophilic colloidal iron stain indicated that the malaria parasite's surface lacked exposed sialic acid groups which would normally give rise to a net negative surface charge common to erythrocytes. Biochemical assay demonstrated that malaria parasites contained about one-half the amount of sialic acid per unit weight as did control red cell extracts. Similarly, external acidic mucopolysaccharide coats of free parasites, as revealed by ruthenium red staining were extremely thin as compared with the thick glycocalyx layer of red cells. Lipid plaques at the surface of parasites and red cells were localized by lipophilic iron colloid staining. Although the gross patchwork distribution of plaques was somewhat similar for the two cell types, the parasites were stained more intensely and had a closer-knit patchwork pattern than those exhibited by the erythrocytes. Such findings indicate that there are slight differences in the arrangement of phospholipids at the surfaces of limiting membranes of host cells and parasites. The significance of the above cytochemical surface properties of the malaria parasite (which are seemingly akin to those of intracellular organelles is discussed in relation to certain host-parasite interactions, such as parasite adhesion to target cells and enhanced clearance of extracellular parasites.     

Plasmodium chabaudi malaria: protective immunization with surface membranes of infected erythrocytes.

Plasmodium chabaudi-susceptible NMRI and B10.A mice were vaccinated with host cell plasma membranes isolated from P. chabaudi-infected erythrocytes. Most of the mice were protected from the lethal consequences of challenge with the homologous parasite, although protection was unassociated with a reduction in the course or peak of parasitemia. Vaccination also induced the production of antibodies against Pc90, which is the immunodominant protein expressed by parasites in host cell plasma membranes.     

Characterization of a Plasmodium falciparum polypeptide associated with membrane vesicles in the infected erythrocytes

A Plasmodium falciparum polypeptide (46 kDa) associated with the infected erythrocytes of all asexual stages as well as immature gametocytes was identified by the monoclonal antibody (Mab) 30B8.3. The expression of this protein was not dependent upon the knobby phenotype and was detected in parasites grown either in human or Aotus erythrocytes. The antigen was heatstable, did not label with [14C]glucosamine, and was not sensitive to periodate oxidation. Immunofluorescent staining patterns of Mab 30B8.3 on in vitro cultured parasites varied from punctate (rings and trophozoites) to patchy (trophozoites and schizonts) fluorescence. The Mab 30B8.3 antigen was not detected on the infected erythrocyte surface by conventional wet-mount IFA procedure. However, when parasites were cultured in the presence of Mab 30B8.3, the epitope was detected by the monoclonal antibodies present in the culture medium. Differential extraction of the polypeptide from infected erythrocytes and immune electron microscopy of cryosectioned parasites localized the 30B8.3 epitope primarily on membranes of Maurer's clefts within the infected erythrocyte's cytosol. This 46 kDa polypeptide is unique because it seemed to be an integral membrane protein of the Maurer's clefts/vesicles and it was not secreted into the culture medium nor deposited on the infected erythrocyte membrane. Previous studies indicate that several parasite proteins, excreted extracellularly or deposited on infected erythrocyte membrane, are found to be associated with Maurer's cleft membranes and vesicles. The 46 kDa polypeptide described in this study may play an important role in the transport of the parasite antigens.     

Glycoprotein recognition mediates attachment of Plasmodium chabaudi to mouse erythrocytes

The interaction between Plasmodium falciparum merozoites and human erythrocytes is mediated by specific parasite proteins and sialoglycoproteins (SGPs) on the surface of the host cell. To investigate whether a similar mechanism functions in rodent malaria, a series of experiments was performed to identify the proteins involved in the interaction of Plasmodium chabaudi parasites and mouse erythrocytes. Labeled parasite proteins incubated with purified mouse SGP bound specifically to glycoprotein 2.1. Two parasite proteins (72 and 126 kilodaltons [kDa]) were coprecipitated with antibody directed to mouse erythrocyte membrane proteins. The lower band (72 kDa) as well as a band of 105 kDa were also observed to bind to N-acetyl-D-galactosamine affinity columns, suggesting a carbohydrate component in the binding of these parasites to erythrocytes. These experiments indicate that P. chabaudi possesses specific proteins which recognized SGP on the surface of murine erythrocytes in a manner similar to that of the merozoites of P. falciparum. Thus P. chabaudi in mice may provide an in vivo model of the human parasite for testing ways to inhibit merozoite recognition and invasion of host cells.     

Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Time-course of synthesis,transport and incorporation of a protein identified in purified membranes of host erythrocytes infected with a knob-forming strain of Plasmodium falciparum

Membranes from host erythrocytes infected with a knob-positive strain of Plasmodium falciparum were purified by free-flow electrophoresis or gradient centrifugation. In these membranes the main parasite-derived protein was a 92 000 Da protein which was not present after infection of erythrocytes with the corresponding knob-negative strain. The protein is synthesized between 9–21 h after merozoite invasion and then synthesis ceases. At least 6 h elapses between the start of synthesis and the appearance of the protein in the erythrocyte membrane. No precursor proteins for the 92 000 Da protein were found. Since the purified erythrocyte membranes were free from contamination with whole parasites or parasite membranes, the 92 000 Da protein is clearly present in the host erythrocyte membrane.     

The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes.

The fluorescent lipophilic probe 1,1'-dihexadecyl-3-3'-3-3'- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. The fluorescent lipid was not transported from red cell to parasite membranes at subsequent stages of development, but label in the erythrocyte-derived parasitophorous vacuole was eventually detected in daughter merozoites. Fluorescent dextrans of 10 kDa in the extracellular medium were also not internalised during intraerythrocytic parasite growth. The results support that with the exception of the invasion step, plasmodial infection does not induce endocytosis in the erythrocyte membrane. Despite the lack of endocytosis, both D and L stereoisomers of the head group blocked phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) inserted in the erythrocyte membrane, were internalised by mature infected erythrocytes. Lipid internalisation occurred by a non head group dependent parasite mechanism, which could account for the stage-specific uptake of phospholipids observed in mature infected erythrocytes. We were unable to detect the transport of carbocyanine dyes and N-NBD-PE from intracellular parasites back to the erythrocyte membrane. Additionally, the carbocyanine dyes were not transferred between adjacent organisms in a double infected red cell. The data argue for the absence of bulk membrane lipid transport between individual parasites and their host cell bilayer in an infected erythrocyte.     

Effect of Kupffer cell phagocytosis of erythrocytes and erythrocyte ghosts on susceptibility to endotoxemia and bacteremia.

The phagocytosis of erythrocytes by macrophages has previously been shown to depress macrophage function. In this study we compared the effect of the phagocytosis of erythrocytes and erythrocyte ghosts by Kupffer cells on the duration of the depression of complement receptor clearance function and host defense against endotoxemia and bacteremia. Phagocytosis of erythrocytes and erythrocyte ghosts was induced in rats by the injection of rat erythrocytes or erythrocyte ghosts coated with anti-rat erythrocyte immunoglobulin G (EIgG and GIgG, respectively). The hepatic uptake of EIgG and GIgG (17.4 X 10(8)/100 g) occurred during the first 30 min after injection. The digestion of phagocytized EIgG and GIgG, as assessed by electron microscopy, was complete at 24 and 3 h after injection, respectively. The depression of Kupffer cell complement receptor clearance function caused by EIgG and GIgG returned to normal by 6 h after injection of EIgG and by 3 h after injection of GIgG. Phagocytosis of EIgG depressed the survival rate after endotoxemia and bacteremia when endotoxin or bacteria were injected at 30 min after EIgG. The survival rate returned to normal when the endotoxin and bacteria were injected at 12 and 6 h after the EIgG, respectively. Phagocytosis of GIgG did not depress the survival rate after endotoxemia and bacteremia. Thus, compared with erythrocytes, erythrocyte ghosts are more rapidly digested after phagocytosis, depress complement receptor function for a shorter period of time, and cause less depression of host defense. These findings indicate that the contents of erythrocytes play an important role in the impairment of host defense caused by the phagocytosis of erythrocytes by Kupffer cells.     

Identification and localization of an adhesin on the surface of Tritrichomonas foetus

Tritrichomonas foetus is a mucosal parasite of the urogenital-vaginal tract of cattle that strongly adheres to erythrocytes, which suggests that it presents an adhesin that recognizes red blood cells from different animal species and blood groups. In the present report we describe a cell-fractionation method for obtainment of a membrane fraction of T. foetus, which adhered to red blood cells. The T. foetus adhesin was obtained after parasite lysis and fractionation followed by ultracentrifugation, whereby a 100,000-g pellet fraction showed a strong hemagglutinating activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction, of erythrocyte ghosts, and of ghosts allowed to interact with the parasite membrane fraction revealed the presence of a 100-kDa protein as the putative adhesin. Polyclonal antibodies obtained in rabbits immunized with this protein recognized proteins of 100 and 90 kDa as determined by immunoblotting. Confocal laser scanning microscopy and transmission electron microscopy of cells incubated first in the presence of the antibody and subsequently in the presence of fluorescein- or gold-labeled goat anti-rabbit IgG showed labeling of the protozoan surface as well as of some cytoplasmic vesicles.     

The formation of heinz bodies in ghosts of human erythrocytes of adults and newborn infants

Summary Isolated membranes (ghosts) of human erythrocytes of newborn infants produce Heinz bodies faster after an exposure to acetylphenylhydrazine than the ghosts of adults. Heinz bodies form faster in ghosts prepared from erythrocytes of newborn infants, incubated in their own plasma or in the plasma of adults, than in ghosts of incubated red cells of adults. A direct relation can be seen between the hemoglobin content of the ghosts and the formation of Heinz bodies. It is suggested that the Heinz body susceptibility of normal human erythrocytes depends on the quantity of hemoglobin bound to the red cell membrane.This work was supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Grants No. Schr 86/8 and Schr 86/9.     

Plasmodium falciparum-infected erythrocytes contain an adenylate cyclase with properties which differ from those of the host enzyme

It has been postulated that differentiation of the human malaria parasite, Plasmodium falciparum, is controlled by cAMP levels. We have determined that P. falciparum synthesizes an adenylate cyclase with several properties distinct from those of the mammalian host cell enzyme. Adenylate cyclase activity was compared in P. falciparum-infected erythrocytes, isolated parasites free of host cell material, and uninfected erythrocyte membranes. The parasite enzyme was unaffected by GTP gamma S, AlF4-, and forskolin, while the erythrocyte enzyme was markedly stimulated by each of these compounds. The parasite adenylate cyclase also exhibited a striking preference for Mn2+ over Mg2+, which was not evident in the erythrocyte enzyme. Moreover, differing cation and pH sensitivities were observed for adenylate cyclase activity in the two cell types. When infected and uninfected erythrocytes were compared, the basal adenylate cyclase activity of infected cells was 7 and 49 times that measured in uninfected erythrocytes in the presence of Mg2+ and Mn2+, respectively. Furthermore, adenylate cyclase activity in infected cells exhibited properties typical of the parasite enzyme. This indicates that synthesis of the parasite enzyme rather than stimulation of the host enzyme accounts for the increased activity in infected cells.     

Cryo scanning electron microscopy of Plasmodium falciparum‐infected erythrocytes

Plasmodium falciparum invades erythrocytes as an essential part of their life cycle. While living inside erythrocytes, the parasite remodels the cell's intracellular organization as well as its outer surface. Late trophozoite‐stage parasites and schizonts introduce numerous small protrusions on the erythrocyte surface, called knobs. Current methods for studying these knobs include atomic force microscopy and electron microscopy. Standard electron microscopy methods rely on chemical fixation and dehydration modifying cell size. Here, a novel method is presented using rapid freezing and scanning electron microscopy under cryogenic conditions allowing for high resolution and magnification of erythrocytes. This novel technique can be used for precise estimates of knob density and for studies on cytoadhesion.     

Phospholipids in parasitic protozoa

Parasitic protozoa are surrounded by membrane structures that have a different lipid and protein composition relative to membranes of the host. The parasite membranes are essential structurally and also for parasite specific processes, like host cell invasion, nutrient acquisition or protection against the host immune system. Furthermore, intracellular parasites can modulate membranes of their host, and trafficking of membrane components occurs between host membranes and those of the intracellular parasite. Phospholipids are major membrane components and, although many parasites scavenge these phospholipids from their host, most parasites also synthesise phospholipids de novo, or modify a large part of the scavenged phospholipids. It was recently shown that some parasites like Plasmodium have unique phospholipid metabolic pathways. This review will focus on new developments in research on phospholipid metabolism of parasitic protozoa in relation to parasite-specific membrane structures and function, as well as on several targets for interference with the parasite phospholipid metabolism with a view to developing new anti-parasitic drugs.     

Scanning electron microscopy in the investigation of the in vitro hemolytic activity of Trichomonas vaginalis

The in vitro hemolytic activity of Trichomonas vaginalis has been previously demonstrated, but the mechanisms involved remain to be elucidated. In this work we used scanning electron microscopy to investigate the contact dependency of the hemolytic phenomenon caused by the parasites. The erythrocytes adhered to the parasites' surface and were phagocytosed. These observations suggest that the contact between T. vaginalis and erythrocytes may be an important mechanism in the injury caused to the erythrocytes. The hemolytic activity of T. vaginalis may be an efficient means of obtaining nutrients for the parasite and allow the investigation of the mechanism used by T. vaginalis to damage cellular membranes.     

Rapid transport of the acidic phosphoproteins ofPlasmodium berghei andP. chabaudi from the intraerythrocytic parasite to the host membrane using a miniaturized fractionation procedure

A miniaturized procedure for the separation of the host erythrocyte membrane from malarial parasites based on saponin lysis and density-gradient centrifugation with Percoll is described. The procedure requires only 20–35 l packed infected erythrocytes, is simple to perform, needs no sophisticated equipment, and can be completed in <2 h. Analysis of the isolated erythrocyte membranes and parasites using marker enzymes and electron microsropy revealed that both the purity and the yield of these fractions were relatively high. Erythrocyte membrane proteins, including spectrin, ankyrin, and band 4.1, were not found on the parasitophorous vacuolar membrane, which remained associated with some but not all of the isolated parasites. Application of this method to pulse-chase experiments indicated that the acidic phosphoproteins ofPlasmodium berghei andP. chabaudi were rapidly transported from the parasite to the erythrocyte membrane immediately after their synthesis. The rapid export of these acidic phosphoproteins from the parasite distinguishes them from other proteins exported by the malarial parasite.     

Identification and purification of glucose phosphate isomerase of Plasmodium falciparum.

The multiplication of malaria parasites within red blood cells is energy dependent. Since these parasites lack a functional tricarboxylic acid cycle, the energy needs of the parasite are met by anaerobic glycolysis of exogenous glucose. High levels of glycolytic enzymes such as fructose-1,6-diphosphate aldolase, phosphoglycerate kinase and pyruvate kinase have been detected in infected erythrocytes. Here we report a 4-9 times increase in glucose phosphate isomerase (GPI) activity of infected erythrocytes over that of normal erythrocytes. This increase is of parasitic origin, as additional enzyme bands were observed in lysates of infected erythrocytes. The expression of GPI parallels parasite maturation and reaches a maximum at the trophozoite/schizont stage. Two distinct but closely related activity patterns consisting of 3-4 GPI isoenzymes (not shown in normal erythrocytes) with neutral to weakly acidic isoelectric points were observed in 6 P. falciparum isolates tested by isoelectric focusing. The purified P. falciparum GPI has an apparent size of 66 kDa. No size variation was observed in the 6 P. falciparum isolates studied. Furthermore, antiserum raised against this protein in BALB/c mice specifically inhibits parasite encoded GPI activity while no effect was observed on host enzyme activity.