Thrombospondin binds falciparum malaria parasitized erythrocytes and may mediate cytoadherence

Plasmodium falciparum infected erythrocytes containing mature trophozoites and schizonts sequester along venular endothelium and are not in the peripheral circulation of patients with malaria. Knobs appear on infected erythrocytes and are the points of attachment to endothelium. Sequestration may protect the parasite from splenic destruction and may play a role in the pathogenesis of cerebral malaria. Correlates of sequestration have been developed in vitro using cultured human endothelium and an amelanotic melanoma cell line. Knobless strains (K-) of P. falciparum fail to sequester in vivo and to bind to cells in vitro. We now present evidence that the receptor for cytoadherence is the glycoprotein, thrombospondin. Aotus monkey or human erythrocytes containing knobby (K+) but not Aotus erythrocytes containing knobless strains of P. falciparum bind to immobilized thrombospondin. Neither binds to the adhesive proteins laminin, fibronectin, factor VIII/von Willebrand factor or vitronectin. Both soluble thrombospondin and anti-thrombospondin antibodies inhibit binding of parasitized Aotus erythrocytes to immobilize thrombospondin and to melanoma cells which secrete thrombospondin.     

Frequent ectopic recombination of virulence factor genes in telomeric chromosome clusters of P. falciparum

Persistent and recurrent infections by Plasmodium falciparum malaria parasites result from the ability of the parasite to undergo antigenic variation and evade host immune attack. P. falciparum parasites generate high levels of variability in gene families that comprise virulence determinants of cytoadherence and antigenic variation, such as the var genes. These genes encode the major variable parasite protein (PfEMP-1), and are expressed in a mutually exclusive manner at the surface of the erythrocyte infected by P. falciparum. Here we identify a mechanism by which var gene sequences undergo recombination at frequencies much higher than those expected from homologous crossover events alone. These recombination events occur between subtelomeric regions of heterologous chromosomes, which associate in clusters near the nuclear periphery in asexual blood-stage parasites or in bouquet-like configurations near one pole of the elongated nuclei in sexual parasite forms. We propose that the alignment of var genes in heterologous chromosomes facilitates gene conversion and promotes the diversity of antigenic and adhesive phenotypes. The association of virulence factors with a specific nuclear subcompartment may also have implications for variation during mitotic recombination in asexual blood stages.     

A chromosomal rearrangement in a P. falciparum histidine-rich protein gene is associated with the knobless phenotype

The significant morbidity and mortality associated with Plasmodium falciparum malaria results, in part, from the sequestration of parasitized erythrocytes in postcapillary venules, which may protect the parasite from splenic clearance and contribute to the pathogenesis of cerebral malaria. This sequestration has been linked to the expression of parasite-induced knob structures on the surface of the infected erythrocyte which mediate the cytoadherence phenomenon. While knobs are necessary for cytoadherence, they are not sufficient, requiring both parasite- and host-encoded proteins. Spontaneous mutants of P. falciparum have been isolated from in vitro cultures which lack the ability to express knobs and fail to cytoadhere. A histidine-rich protein has been described which is associated with the knobby phenotype and may be a constituent of the knob. We now report the isolation of complementary DNA clones for a knob-associated histidine-rich protein (KAHRP) and demonstrate that in knobless mutants the gene for this protein has undergone a rearrangement, resulting in a deletion in the 3' coding sequence. Moreover, the chromosome to which the KAHRP gene maps is rearranged in these mutants, producing a telomeric location of the truncated gene. These observations explain the loss of expression of the messenger RNA and protein in such mutants and may explain the loss of the knob itself. The implications for the generation of spontaneous mutations in the parasite by this novel mechanism are discussed.     

Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells.

The malaria parasite Plasmodium falciparum is one of the most successful human pathogens. Specific virulence factors remain poorly defined, although the adhesion of infected erythrocytes to the venular endothelium has been associated with some of the syndromes of severe disease. Immune responses cannot prevent the development of symptomatic infections throughout life, and clinical immunity to the disease develops only slowly during childhood. An understanding of the obstacles to the development of protective immunity is crucial for developing rational approaches to prevent the disease. Here we show that intact malaria-infected erythrocytes adhere to dendritic cells, inhibit the maturation of dendritic cells and subsequently reduce their capacity to stimulate T cells. These data demonstrate both a novel mechanism by which malaria parasites induce immune dysregulation and a functional role beyond endothelial adhesion for the adhesive phenotypes expressed at the surface of infected erythrocytes.     

Abnormal display of PfEMP-1 on erythrocytes carrying haemoglobin C may protect against malaria

Haemoglobin C, which carries a glutamate-to-lysine mutation in the beta-globin chain, protects West African children against Plasmodium falciparum malaria. Mechanisms of protection are not established for the heterozygous (haemoglobin AC) or homozygous (haemoglobin CC) states. Here we report a marked effect of haemoglobin C on the cell-surface properties of P. falciparum-infected erythrocytes involved in pathogenesis. Relative to parasite-infected normal erythrocytes (haemoglobin AA), parasitized AC and CC erythrocytes show reduced adhesion to endothelial monolayers expressing CD36 and intercellular adhesion molecule-1 (ICAM-1). They also show impaired rosetting interactions with non-parasitized erythrocytes, and reduced agglutination in the presence of pooled sera from malaria-immune adults. Abnormal cell-surface display of the main variable cytoadherence ligand, PfEMP-1 (P. falciparum erythrocyte membrane protein-1), correlates with these findings. The abnormalities in PfEMP-1 display are associated with markers of erythrocyte senescence, and are greater in CC than in AC erythrocytes. Haemoglobin C might protect against malaria by reducing PfEMP-1-mediated adherence of parasitized erythrocytes, thereby mitigating the effects of their sequestration in the microvasculature.     

Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody

Red blood cells infected with mature stages of the malaria parasite Plasmodium falciparum bind to the endothelial lining of capillaries and venules. This sequestration is important for the survival of the parasite but may have severe consequences for the host. For example, it is involved in the causation of cerebral malaria which carries 25% mortality. Knob-like protrusions present on the surface of infected erythrocytes have been considered necessary but not sufficient for this cytoadherence. Here we describe the adhesion to endothelial cells of infected erythrocytes which do not have knobs. A human monoclonal antibody (33G2) which was specific for an epitope containing regularly spaced dimers of glutamic acid present in the repeated amino-acid sequences of some defined P. falciparum antigens was found to inhibit cyto-adherence and may therefore be an important reagent for elucidating the molecular basis of parasite sequestration.     

Structural basis for Duffy recognition by the malaria parasite Duffy-binding-like domain

Molecular processes that govern pathogenic features of erythrocyte invasion and cytoadherence in malaria are reliant on Plasmodium-specific Duffy-binding-like domains (DBLs). These cysteine-rich modules recognize diverse host cell-surface receptors during pathogenesis. DBLs of parasite erythrocyte-binding proteins mediate invasion, and those from the antigenically variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) have been implicated in cytoadherence. The simian and human malarial parasites, P. knowlesi and P. vivax, invade human erythrocytes exclusively through the host DARC receptor (Duffy antigen receptor for chemokines). Here we present the crystal structure of the P. knowlesi DBL domain (Pkalpha-DBL), which binds to DARC during invasion of human erythrocytes. Pkalpha-DBL retains the overall fold observed in DBLs from P. falciparum erythrocyte-binding antigen (EBA)-175 (ref. 4). Mapping the residues that have previously been implicated in binding highlights a fairly flat but exposed site for DARC recognition in subdomain 2 of Pkalpha-DBL; this is in sharp contrast to receptor recognition by EBA-175 (ref. 4). In Pkalpha-DBL, the residues that contact DARC and the clusters of residues under immune pressure map to opposite surfaces of the DBL, and suggest a possible mechanism for immune evasion by P. vivax. Our comparative structural analysis of Pkalpha-DBL and P. falciparum EBA-175 provides a framework for the understanding of malaria parasite DBLs, and may affect the development of new prophylactic and therapeutic strategies.     

Plasmodium falciparum strain-specific antibody blocks binding of infected erythrocytes to amelanotic melanoma cells

An important feature of Plasmodium falciparum malaria which differentiates it from other human malarias is that erythrocytes infected with trophozoites and schizonts are not present in the peripheral blood but are sequestered along capillary and venular endothelium. Infected erythrocytes attach via parasite-induced ultrastructural modifications on the surface of the infected cells, called 'knobs'. This sequestration may be important for parasite survival because it prevents infected erythrocytes from circulating through the spleen where they could be eliminated. We have established an in vitro correlate of sequestration and used it to demonstrate that immune sera from repeatedly infected Aotus monkeys inhibit binding of infected erythrocytes to endothelial cells. We have investigated whether antiserum that blocks binding of one isolate of P. falciparum to target cells can block or reverse binding of other isolates. We report here that sera which block or reverse binding are strain-specific, indicating that the corresponding antigens on the surface of the infected erythrocytes are strain (isolate)-specific.     

A protein on Plasmodium falciparum-infected erythrocytes functions as a transferrin receptor

Several observations suggest that iron is essential for the development of malaria parasites but there is evidence that the parasites in erythrocytes do not obtain iron from haemoglobin. The total haemin level in parasitized erythrocytes does not vary during parasite development, indicating that the iron-containing moiety of haemoglobin is not detectably metabolized. Although parasite proteases can degrade the protein part of haemoglobin in red cells, no parasite enzymes that degrade haemin have been identified. In mammalian cells, haemin is degraded to carbon monoxide and bilirubin by the enzyme haeme oxygenase. This enzyme has not been found in malaria parasites. In fact haemin has been found to be toxic to parasite carbohydrate metabolism. Thus, iron apparently cannot be liberated from haemin and instead is sequestered in infected red cells as haemozoin, the characteristic pigment associated with malarial infection. If iron bound to transferrin is the source of ferric ions for malaria parasites within mature erythrocytes, then the parasite must synthesize its own transferrin receptor and localize it on the surface of the infected cell, because the receptors for transferrin are lost during erythrocyte maturation. Our results here suggest that Plasmodium falciparum synthesizes its own transferrin receptors enabling it to take up iron from transferrin by receptor-mediated endocytosis.     

Immune sera recognize on erythrocytes Plasmodium falciparum antigen composed of repeated amino acid sequences

Protective immune responses against the asexual stages of the human malaria parasite, Plasmodium falciparum, are most probably directed against exposed antigenic determinants on the surface of the free merozoite or the infected red blood cell, and therefore antigens in these locations are candidates for testing as components of a defined molecular vaccine. To facilitate the search for such antigens, we recently developed a method for the expression of P. falciparum proteins in Escherichia coli as fused polypeptides. Many clones producing antigens were detected by screening with immune human sera. We show here that antibodies against the fused polypeptide expressed by one such clone react with a P. falciparum protein that is synthesized late in schizogony and is later present on the surface of the ring-infected erythrocyte. The protein is composed of repeating subunits of 8, 4 and 3 amino acids and is present in all isolates of P. falciparum examined.     

Transient cross-reactive immune responses can orchestrate antigenic variation in malaria

The malaria parasite Plasmodium falciparum has evolved to prolong its duration of infection by antigenic variation of a major immune target on the surface of the infected red blood cell. This immune evasion strategy depends on the sequential, rather than simultaneous, appearance of immunologically distinct variants. Although the molecular mechanisms by which a single organism switches between variants are known in part, it remains unclear how an entire population of parasites within the host can synchronize expression to avoid rapidly exhausting the variant repertoire. Here we show that short-lived, partially cross-reactive immune responses to parasite-infected erythrocyte surface antigens can produce a cascade of sequentially dominant antigenic variants, each of which is the most immunologically distinct from its preceding types. This model reconciles several previously unexplained and apparently conflicting epidemiological observations by demonstrating that individuals with stronger cross-reactive immune responses can, paradoxically, be more likely to sustain chronic infections. Antigenic variation has always been seen as an adaptation of the parasite to evade host defence: we show that the coordination necessary for the success of this strategy might be provided by the host.     

硫酸软骨素A介导的恶性疟原虫感染的红细胞粘附

对硫酸软骨素的粘附机制进行了初步探讨。研究表明,恶性疟原虫感染的红细胞可粘附于各种器官的微血管内皮细胞,这种粘附被认为是红细胞膜表面分子与内皮细胞表面分子间配体－受体相互作用的结果,ＣＳＡ为内皮细胞表面感染的红细胞的粘附受体和恶性疟原虫红细胞膜蛋白１的配体。     

Inhibition of P. falciparum growth in human erythrocytes by monoclonal antibodies

Malaria is increasing in incidence and prevalence in most tropical areas and is a major problem for both individuals and communities. Current malaria research is aimed at developing vaccines and, for this, it may be useful to define Plasmodium antigen(s) related to the development of a protective immune response in the host. Monoclonal antibodies have recently been shown to interfere with rodent malaria infection (Plasmodium berghei) at the sporozoite or merozoite stage. We have now raised monoclonal antibodies against single antigenic determinant(s) of Plasmodium falciparum and report that some of them inhibit the growth of erythrocytic forms of P. falciparum in vitro.     

Evidence for immunological cross-reaction between sporozoites and blood stages of a human malaria parasite

Malaria parasites (Plasmodium spp.) show a complex pattern of development in the mammalian host and many studies support the view that the surface of the sporozoite, injected by the mosquito, has no antigens in common with the erythrocytic stage of development. For example, immunization with the erythrocytic parasites generates antisera with negligible titre by indirect immunofluorescence to the sporozoite surface. Although monoclonal antibodies prepared against erythrocytic stages were reported to show cross-reaction to the sporozoite stage, this appeared to be due to cytoplasmic antigens exposed by the method of sporozoite preparation, and in Plasmodium knowlesi, a cDNA clone coding for the circumsporozoite antigen, the major protein of the sporozoite surface, showed no hydridization to mRNA isolated from the erythrocytic stages. Here, however, we present evidence for an antigenic determinant shared by the sporozoite surface and the erythrocytic stages of the human malaria parasite, P. falciparum. Moreover, our studies show that the antigen(s) elicit a strong immune response in man.     

IgM-autoantibodies against isologous erythrocytes also react with isologous IgG(Fc)

The mechanisms by which the adaptive immune system of animals discriminate between foreign and self components are not known. It is generally believed that antibodies are produced only in response to foreign agents. However, against this axiom there is mounting evidence that normal animals produce autoantibodies against a variety of self components. In two murine model systems, a high proportion of IgM-producing cells secrete autoantibodies. In one model, the autoantibodies are specific for isologous mouse IgG(Fc) and in the other, the autoantibodies are specific for antigens revealed on the surface of mouse erythrocytes (RBC) by proteolytic enzymes. The function of these two autoimmune responses is a mystery. Here we show that the two responses are related. The lysis of mouse RBC, modified with bromelain (brom) by IgM autoantibodies was completely inhibited by isologous IgG(Fc), and antibodies against mouse IgG(Fc) formed precipitin bands with solubilized membranes from mouse RBC. We conclude that mouse RBC membranes and IgG(Fc) have at least one antigenic determinant in common and that this determinant is the target for autoimmune responses in normal mice.     

A proteomic view of the Plasmodium falciparum life cycle

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.     

Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum

The primary event in the pathogenesis of severe malaria in Plasmodium falciparum infection is thought to be adherence of trophozoite- and schizont-infected erythrocytes to capillary endothelium, a process called sequestration. Identifying the endothelial molecules used as receptors is an essential step in understanding this disease process. Recent work implicates the membrane glycoprotein CD36 (platelet glycoprotein IV; refs 2-5) and the multi-functional glycoprotein thrombospondin as receptors. Although CD36 has a widespread distribution on microvascular endothelium, it may not be expressed on all capillary beds where sequestration occurs, especially in the brain. The role of thrombospondin in cell adhesion, in vitro or in vivo, is less certain. We have noticed that some parasites bind to human umbilical-vein endothelial cells independently of CD36 or thrombospondin. To screen for alternative receptors, we have developed a novel cell-adhesion assay using transfected COS cells, which confirms that CD36 is a cell-adhesion receptor. In addition, we find that an endothelial-binding line of P. falciparum binds to COS cells transfected with a complementary DNA encoding intercellular adhesion molecule-1. As this molecule is widely distributed on capillaries and is inducible, this finding may be relevant to the pathogenesis of severe malaria.     

Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum

Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor-ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor-ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Ok(a-) erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor-ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.     

Intragenic recombination leads to pilus antigenic variation in Neisseria gonorrhoeae

The pilus of the bacterium Neisseria gonorrhoeae is a fimbriate surface structure which promotes attachment of the bacterium to host epithelial cells. Gonococcal pilus phase variation is characterized by a rapid on/off switch in which piliated (P+) cells throw off non-piliated (P-) variants and vice versa. Two regions of the gonococcal chromosome (pilE1 and pilE2) act as pilin expression loci, reminiscent of the MAT locus in the yeast Saccharomyces cerevisiae, while several other chromosomal regions contain silent (non-expressing) pilin sequences. Biochemical and antigenic diversity is seen in pili from a wide variety of clinical isolates. Pilins (pilus subunits) are composed of conserved N-terminal and variable C-terminal regions; the conserved region of gonococcal pilin is also found in pilins produced by widely disparate bacteria. We show here that the gonococcal pilin undergoes antigenic variation in vitro and in vivo. The protein consists of constant, semi-variable and hypervariable regions. This antigenic variation probably involves gene conversion of mini-cassettes of pilin information.