Isolation and characterization of parasites and host cell ghosts from erythrocytes infected with Plasmodium chabaudi

A new procedure has been developed which allows the concomitant isolation of viable parasites and host cell plasma membranes from erythrocytes infected with Plasmodium chabaudi trophozoites. The average final yield of parasites is 56%. Free parasites reveal a well preserved ultrastructure, incorporate [14C]isoleucine for at least 3 h, and synthesize about the same proteins as parasites within erythrocytes as monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)-autoradiography. The host cell plasma membranes can be isolated in the form of ghosts with an average yield of 27%. The ghosts possess a structurally intact plasma membrane as revealed by freeze-etch electron microscopy. The ghosts are regularly associated with seven neo-proteins as identified by SDS-PAGE and isoelectric focusing (IEF)/SDS-PAGE. These neo-proteins have the following apparent molecular masses: 154 kDa, 145 kDa, 90 kDa, 72 kDa (pI 4.5), 67 kDa, 52 kDa, and 33 kDa (pI 5.7), respectively. The contamination of ghosts by parasite material and, conversely, the contamination of parasites by host cell plasma membranes is very low as demonstrated by light and electron microscopy, lactoperoxidase-mediated radioiodination and the distribution of the typical parasite marker enzymes such as choline kinase, cholinephosphotransferase and ethanolaminephosphotransferase.     

Serological cross-reaction between high molecular weight proteins synthesized in blood schizonts of Plasmodium yoelii, Plasmodium chabaudi and Plasmodium falciparum

A 230 000 molecular weight (MW) Plasmodium yoelii protein, a 250 000 MW P. chabaudi protein and a 195 000 MW P. falciparum protein, identified using monoclonal antibodies, have similar characteristics, and have been implicated as protective antigens. In this study the serological relationship between these proteins was investigated by Western transfer analysis. The monoclonal antibodies specific for each of the high molecular weight proteins did not cross-react with antigens of the other two parasites, but a polyvalent mouse serum raised against the purified 230 000 MW P. yoelii protein cross-reacted with the high molecular weight proteins of P. chabaudi and P. falciparum and also with the fragments derived from these proteins. This result indicates that these proteins belong to the same class of malaria parasite antigen.     

Antibody recognition of rodent malaria parasite antigens exposed at the infected erythrocyte surface: specificity of immunity generated in hyperimmune mice

In regions where malaria is endemic, inhabitants remain susceptible to repeated reinfection as they develop and maintain clinical immunity. This immunity includes responses to surface-exposed antigens on Plasmodium sp.-infected erythrocytes. Some of these parasite-encoded antigens may be diverse and phenotypically variable, and the ability to respond to this diversity and variability is an important component of acquired immunity. Characterizing the relative specificities of antibody responses during the acquisition of immunity and in hyperimmune individuals is thus an important adjunct to vaccine research. This is logistically difficult to do in the field but is relatively easily carried out in animal models. Infections in inbred mice with rodent malaria parasite Plasmodium chabaudi chabaudi AS represent a good model for Plasmodium falciparum in humans. This model has been used in the present study in a comparative analysis of cross-reactive and specific immune responses in rodent malaria. CBA/Ca mice were rendered hyperimmune to P. chabaudi chabaudi (AS or CB lines) or Plasmodium berghei (KSP-11 line) by repeated infection with homologous parasites. Serum from P. chabaudi chabaudi AS hyperimmune mice reacted with antigens released from disrupted P. chabaudi chabaudi AS-infected erythrocytes, but P. chabaudi chabaudi CB and P. berghei KSP-11 hyperimmune serum also contained cross-reactive antibodies to these antigens. However, antibody activity directed against antigens exposed at the surfaces of intact P. chabaudi chabaudi-infected erythrocytes was mainly parasite species specific and, to a lesser extent, parasite line specific. Importantly, this response included opsonizing antibodies, which bound to infected erythrocytes, leading to their phagocytosis and destruction by macrophages. The results are discussed in the context of the role that antibodies to both variable and invariant antigens may play in protective immunity in the face of continuous susceptibility to reinfection.     

Identification of a Plasmodium chabaudi antigen present in the membrane of ring stage infected erythrocytes

A novel antigen of asexual blood stages of the rodent malaria parasite Plasmodium chabaudi, was detected by means of a modified indirect immunofluorescence assay (IFA), using glutaraldehyde fixed and air dried monolayers of P. chabaudi infected erythrocytes. P. chabaudi hyperimmune sera gave a distinct surface immunofluorescence of erythrocytes infected with early stages of the parasite. Fixation and drying of the erythrocytes was necessary for the antigenic activity to be exposed. The antigens were species specific as P. chabaudi hyperimmune serum only stained P. chabaudi but not P. yoelii or P. falciparum infected erythrocytes. The antigenic activity involved in the IFA was resistant to trypsin, phospholipases and neuraminidase but not to pronase, suggesting that the antigens were polypeptides. The surface immunofluorescence was inhibited by an extract of parasitized erythrocytes, but not by similar extracts of normal erythrocytes. The inhibitory antigens were soluble and heat stable (100 degrees C, 5 min). For identification and characterization of the antigens, antibodies were isolated by acid elution from monolayers of infected erythrocytes and monoclonal antibodies were produced. Probing in immunoblotting of extracts of parasitized erythrocytes separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis with the eluted antibodies, showed that they reacted consistently with a polypeptide of Mr 105 000 (Pch105). The Pch105 antigen shares many characteristics with Pf155, a P. falciparum antigen considered as a candidate for a vaccine against that parasite.     

TNF and inhibition of growth of Plasmodium falciparum

The mechanism of intra-erythrocyte death of Plasmodium chabaudi in vivo has not yet been elucidated. Here we summarise recent experiments in which serum from mice undergoing a successful immune response to this parasite did not inhibit Plasmodium falciparum in vivo unless the P. chabaudi infection and TNF levels were high enough to cause illness in the host. This was true for the 556KA and DS strains of P. chabaudi in intact mice, but not for 556KA in nude mice, which did not generate inhibitory activity at any parasitaemia. Tumour necrosis factor (TNF) inhibits malaria parasites via some undefined secondary mediator. 10 mg of r hu TNF generated this inhibitory activity, as measured against P. falciparum in vitro, in the serum of mice only if they were pretreated with Corynebacterium parvum, which activates macrophages and sensitises the mice to the toxic effects of TNF. This implies a role for activated macrophages downstream from TNF in the process involved in intra-erythrocytic death of parasites.     

Acidic phosphoproteins associated with the host erythrocyte membrane of erythrocytes infected with Plasmodium berghei and P. chabaudi

New phosphoproteins appear on the host erythrocyte membrane during Plasmodium berghei and P. chabaudi infection. Distinct proteins having similar properties and all distinguished by isoelectric points of less than 4.0 are identified. Associated with the erythrocyte membranes of P. berghei infected erythrocytes are two proteins with molecular masses of 65 and 46 kDa, whereas 93, 90 and 76 kDa proteins are observed during P. chabaudi infection. These new erythrocyte membrane associated proteins are all of parasite origin as indicated by metabolic labeling with proline and are synthesized during the ring stage of the asexual replicative cycle. Three of these proteins, the 93 kDa P. chabaudi protein and both P. berghei proteins, have been purified and the amino acid composition determined. All three are characterized by a relatively high proportion of aspartate and glutamate residues. Mono-and polyclonal antibodies were also raised against the same three purified proteins. No cross reactivity between these three proteins is observed, but one monoclonal antibody against the 65 kDa P. berghei crossreacts with a 27 kDa mouse erythrocyte protein. Immunofluorescence using the antibodies in combination with subcellular fractionation studies clearly shows that these phosphoproteins are associated with the host erythrocyte membrane and not the parasite.     

Plasmodium chabaudi-infected erythrocytes adhere to CD36 and bind to microvascular endothelial cells in an organ-specific way

Adherence of erythrocytes infected with Plasmodium falciparum to microvascular endothelial cells (sequestration) is considered to play an important role in parasite virulence and pathogenesis. However, the real importance of sequestration for infection and disease has never been fully assessed. The absence of an appropriate in vivo model for sequestration has been a major barrier. We have examined the rodent malaria parasite Plasmodium chabaudi chabaudi AS in mice as a potential model. Erythrocytes infected with this parasite adhere in vitro to purified CD36, a critical endothelium receptor for binding P. falciparum-infected erythrocytes. P. c. chabaudi-infected erythrocytes adhere in vitro to endothelial cells in a gamma interferon-dependent manner, suggesting the involvement of additional adhesion molecules in the binding process, as is also the case with P. falciparum-infected cells. Furthermore, plasma or sera from infected and hyperimmune mice, respectively, have the ability to block binding of infected erythrocytes to endothelial cells. In vivo, erythrocytes containing mature P. c. chabaudi parasites are sequestered from the peripheral circulation. Sequestration is organ specific, occurring primarily in the liver, although intimate contact between infected erythrocytes and endothelial cells is also observed in the spleen and brain. The results are discussed in the context of the use of this model to study (i) the relationship between endothelial cell activation and the level of sequestration and (ii) the primary function of sequestration in malaria infection.     

Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion

Rhoptry proteins of Plasmodium falciparum merozoites, of 140, 130, and 110 kDa, identified by co-precipitation with Mab.1B9, bind selectively to mouse erythrocytes and reticulocytes. The properties of binding are shown to correlate with invasion of P. falciparum into mouse erythrocytes. Invasion of two strains of P. falciparum 7G8 and FCR-3, into mouse erythrocytes was examined, and was found to differ significantly. The 7G8 strain invades mouse erythrocytes at a rate of 40-60% compared to invasion into human erythrocytes, whereas FCR-3 invades at a rate of 5-15%. Both strains of P. falciparum preferentially invade reticulocytes in the in vitro invasion assay. This correlated with an increase in the amount of rhoptry protein of the 7G8 strain bound to mouse erythrocytes, compared to the FCR-3 strain and an increased binding to reticulocytes compared to mature erythrocytes. Binding of the rhoptry proteins and merozoite invasion into the erythrocyte is blocked in erythrocytes treated with trypsin and chymotrypsin but not in neuraminidase-treated erythrocytes, suggesting that the putative receptor site is exposed and accessible on the erythrocyte surface. Rabbit antiserum against gp3, the major glycophorin of mouse erythrocytes, blocks binding of the rhoptry proteins to erythrocytes and reduces merozoite invasion into mouse erythrocytes by 50%. Binding of rhoptry proteins to mouse reticulocytes was not blocked by alpha gp3 indicating a receptor difference between reticulocytes and erythrocytes. Mab.1B9 reduces merozoite invasion but does not decrease binding of the rhoptry proteins to the mouse erythrocyte. The mouse erythrocyte serves as a useful model to study the receptor-ligand interaction of rhoptry proteins and host surface proteins and to define the role of the rhoptry proteins during the invasion process.     

The domain on the mouse Duffy protein for Plasmodium yoelii binding and invasion to mouse erythrocytes

Erythrocyte invasion by malaria parasites is a multi-step process requiring specific molecular interactions between merozoites and erythrocyte surface receptors. Human Duffy blood group protein is the receptor for Plasmodium vivax merozoite invasion to red blood cells. The cognate parasite ligand for Duffy protein is a 135 kDa Duffy binding protein (DBP). Previously, we defined the domain on the N-terminus of human Duffy protein required for DBP binding and showed that a 35-mer N-terminal peptide inhibited DBP binding to Duffy positive red cells in vitro. There is no efficient in vitro culture system or small animal model to study P. vivax ligand binding and invasion to red blood cells. Plasmodium yoelii is frequently used to study the interaction between host receptors and parasite ligands. Similar to human parasite P. vivax, rodent malaria parasite P. yoelii also uses Duffy protein on mouse RBCs for invasion. However, the domain on the mouse Duffy for P. yoelii binding is not known. In this communication, using a mouse model, we show that an antibody against the N-terminus of mouse Duffy protein inhibited P. yoelii invasion in the mouse. In addition, by using small peptides from the N-terminal exocellular domain, we defined the domain on the Duffy protein for P. yoelii binding and invasion to mouse erythrocytes. Our results also indicated that small peptides from the host receptor could act as decoy receptors and may be utilized as potential antimalarial drugs.     

Pfsbp1, a Maurer's cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton

Antibodies from hyperimmune monkey sera, selected by absorption to Plasmodium falciparum-infected erythrocytes, and elution at acidic pH, allowed us to characterize a novel parasite protein, Pfsbp1 (P. falciparum skeleton binding protein 1). Pfsbp1 is an integral membrane protein of parasite-induced membranous structures associated with the erythrocyte plasma membrane and referred to as Maurer's clefts. The carboxy-terminal domain of Pfsbp1, exposed within the cytoplasm of the host cell, interacts with a 35 kDa erythrocyte skeletal protein and might participate in the binding of the Maurer's clefts to the erythrocyte submembrane skeleton. Antibodies to the carboxy- and amino-terminal domains of Pfsbp1 labelled similar vesicular structures in the cytoplasm of Plasmodium chabaudi and Plasmodium berghei-infected murine erythrocytes, suggesting that the protein is conserved among malaria species, consistent with an important role of Maurer's cleft-like structures in the intraerythrocytic development of malaria parasites.     

Fractionation of mouse malarious blood according to parasite developmental stage, using a Percoll-sorbitol gradient

Asexual intraerythrocytic malarial parasites permeabilize the membrane of their host cell to small monelectrolytes and anions. Since permeabilization increases with parasite maturation, this property has been used previously to fractionate blood infected with Plasmodium falciparum and P. knowlesi according to the developmental stage of the parasite, using Percoll-sorbitol density gradients. We have extended this method to fractionate mouse blood infected with four species of rodent malaria: P. chahaudi, P. vinckei, P. voelii and P. berghei. While the method works in principle in this case, the polyparasitism which characterizes these species prevented explicit separation according to developmental stage. Hence, erythrocytes harbouring several ring-stage parasites appeared in the same fraction which contained cells hosting a single trophozoite, and polyparasitized trophozoites were associated with singly-infected schizont. This observation implies that permeabilization of the host cell membrane results from the integrated metabolic activity of the parasite(s) and is not related to a specific phase of parasite development.     

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.     

Phosphorylation of erythrocyte membrane and cytoskeleton proteins in cells infected with Plasmodium falciparum

Phosphorylation changes in the erythrocyte membrane and cytoskeletal proteins as a consequence of infection by the malarial parasite Plasmodium falciparum were examined. Spectrin, band 3, band 4.1, ankyrin and glycophorin are phosphorylated in normal erythrocytes. As a consequence of invasion by the merozoite, the extracellular stage of the parasite, into 32P-prelabeled normal erythrocytes, all the major 32P-labeled erythrocyte proteins are dephosphorylated. As the parasite develops intracellularly from the immature ring stage to the mature schizont stage, selective phosphorylation of certain host proteins, spectrin, ankyrin and band 3 is observed. Band 4.1 does not appear to incorporate [32P]phosphate at any stage of parasite development. These observed phosphorylation changes may be important in the regulation of the cytoskeletal organization in P. falciparum-infected cells.     

Band 3 clustering promotes the exposure of neoantigens in Plasmodium falciparum-infected erythrocytes

Erythrocytes infected with the human malaria parasite Plasmodium falciparum become structurally and antigenically modified as a consequence of intracellular parasite development. The new antigens that appear on the surface of the infected erythrocyte originate from parasite-encoded proteins and by modification of the erythrocyte membrane protein band 3. Here, we show that anti-peptide antibodies generated against an amino acid sequence (YETFSKLIKIFQDH) of human band 3, and previously identified as mediating adhesion of infected erythrocytes to CD36, recognized P. falciparum-infected erythrocytes. In addition, sera from individuals living in a malaria endemic area (and who are presumably immune) contained immunoglobulins specific for this region of band 3. The anti-peptide antibodies reacted with the surface excrescences (knobs) on falciparum-infected erythrocytes. In uninfected erythrocytes, the band 3 region was cryptic and its exposure on the falciparum-infected erythrocyte surface required clustering of band 3 protein. Thus, a parasite-induced modification of band 3 promotes adhesion and induces antigenic changes in the P. falciparum-infected erythrocyte.     

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.     

Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium

Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653-3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca2+ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca2+ chelator, BAPTA. When parasites were treated with cytoplasmic Ca2+ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH2, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca2+.     

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.     

In vivo selection of populations of Plasmodium chabaudi chabaudi AS resistant to a monoclonal antibody that reacts with the precursor to the major merozoite surface antigen.

Mice bearing a hybridoma secreting a monoclonal antibody (MAb), MAb-3, which significantly delays the onset of a Plasmodium chabaudi chabaudi AS, but not P. chabaudi chabaudi CB, challenge parasitemia in a passive transfer assay and which is specific for the precursor to the major merozoite surface antigen (PMMSA) of P. chabaudi chabaudi AS, were challenged intravenously with 10(3) P. chabaudi chabaudi AS-parasitized erythrocytes. The resultant parasitemia was very similar to that in normal mice except that initially the parasitemia was sometimes slightly delayed. Parasites derived from cryopreserved stabilates isolated from MAb-3 hybridoma mice with an unmodified parasitemia, or with a delayed parasitemia, were found to have lost their susceptibility to MAb-3 in the passive transfer assay. A number of anti-PMMSA MAb were used to immunoprecipitate lysates of parasite populations isolated directly from hybridoma-bearing mice. In some instances and with certain of the MAb, immunoprecipitation patterns were modified, but other isolates were not detectably different when compared with unselected P. chabaudi chabaudi AS parasites. Using a panel of MAb reacting with the PMMSA of P. chabaudi chabaudi AS, immunoprecipitation patterns of parasites derived from cryopreserved stabilates isolated from hybridoma-bearing mice were determined at 2-h intervals through the appropriate part of the parasite maturation cycle. In these derived populations, resistance to MAb-3 was not associated with a change in the immunoprecipitation reaction with the MAb used. These results are discussed in the context of current knowledge of genotypic and phenotypic antigenic diversity of malaria parasites and other protozoa.     

Correct promoter control is needed for trafficking of the ring-infected erythrocyte surface antigen to the host cytosol in transfected malaria parasites

Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependent on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.     

Use of a DNA probe to analyse the dynamics of infection with rodent malaria parasites confirms that parasite clearance during crisis is predominantly strain- and species-specific

A DNA probe, PCsv4.1, isolated from Plasmodium chabaudi chabaudi AS, generates in Southern blotting experiments restriction fragment length polymorphisms specific to a particular rodent malaria parasite line. It was used to develop an assay which allows identification and semi-quantitative compositional analysis of sample parasite populations in which one or more strains, subspecies or species were present. In experiments where mechanisms effecting parasite clearance during crisis were studied, the assay was used to determine the composition of parasite populations present in P. c. chabaudi AS infected mice challenged during crisis with homologous or heterologous parasites. It was thus confirmed that clearance mechanisms during crisis operate in a predominantly specific manner.