恶性疟原虫毒性蛋白PfEMP1的作用与运输

受恶性疟原虫感染的宿主红细胞会进行细胞重建,其中最明显的改变就是受染红细胞表面出现很多疣状突起(knob)。恶性疟红细胞膜相关蛋白1(PfEMP1)是疟原虫产生的毒性蛋白;这种蛋白通过受染红细胞表面的疣状突起被锚定在红细胞表面,介导红细胞与宿主内皮细胞受体结合,从而使恶性疟原虫能够逃避宿主的清除,并因阻塞作用致宿主脏器功能受损。PfEMP1由疟原虫基因组编码产生,通过其自身的一些特殊结构域将PfEMP1输送至包绕恶性疟原虫的纳虫空泡。由于红细胞没有亚细胞器,因此疟原虫必须建立自己的蛋白运输通路方可将虫体蛋白运至宿主细胞表面。受染红细胞胞质出现的茂氏裂褶是一种分泌细胞器,它将毒性蛋白由纳虫空泡运至红细胞膜表面。     

Trafficking of the major virulence factor to the surface of transfected P. falciparum-infected erythrocytes

After invading human red blood cells (RBCs) the malaria parasite Plasmodium falciparum remodels the host cell by trafficking proteins to the RBC compartment. The virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for cytoadherence of infected cells to host endothelial receptors. This protein is exported across the parasite plasma membrane and parasitophorous vacuole membrane and inserted into the RBC membrane. We have used green fluorescent protein chimeras and fluorescence photobleaching experiments to follow PfEMP1 export through the infected RBC. Our data show that a knob-associated histidine-rich protein (KAHRP) N-terminal protein export element appended to the PfEMP1 transmembrane and C-terminal domains was sufficient for efficient trafficking of protein domains to the outside of the P. falciparum-infected RBC. The physical state of the exported proteins suggests trafficking as a complex rather than in vesicles and supports the hypothesis that endogenous PfEMP1 is trafficked in a similar manner. This study identifies the sequences required for expression of proteins to the outside of the P. falciparum-infected RBC membrane.     

Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.     

Membrane-associated electron-dense material of the asexual stages of Plasmodium falciparum: evidence for movement from the intracellular parasite to the erythrocyte membrane

Electron-dense material (EDM) appears at the parasite plasma membrane with trophozoites of several strains of Plasmodium falciparum cultured in vitro. The EDM is also seen associated with unit membrane-bounded Maurer's clefts in K+ P. falciparum-infected erythrocytes. The cytoplasmic clefts lack the EDM with K- parasites. Some EDM have the same density and appearance as the material located under knobs at the erythrocyte membrane. The EDM at the parasite plasma membrane is absent with schizonts when expression of new knobs at the erythrocyte membrane appears to have ceased. This electron microscopic study suggests that the parasite-derived EDM is transported from the parasite plasmalemma to the erythrocyte membrane via Maurer's clefts in the erythrocyte cytoplasm.     

Subtelomeric chromosome deletions in field isolates of Plasmodium falciparum and their relationship to loss of cytoadherence in vitro.

Subtelomeric deletions are responsible for the loss of expression of several Plasmodium falciparum antigens, including the knob-associated histidine-rich protein (KAHRP). Such deletions are detectable by two-dimensional pulsed-field gradient electrophoresis (PFGE) in which the chromosomes separated in dimension 1 are cleaved with Apa I, and the sizes of telomeric fragments are determined in dimension 2. This sensitive technique has enabled us to examine the role of subtelomeric deletions in two aspects of the biology of Plasmodium falciparum. First, we show that similar subtelomeric deletions to those that occur in vitro also occur in field isolates. Second, we demonstrate a correlation between subtelomeric deletions and loss of the phenotype of "cytoadherence" in cultured isolates. Subclones were generated from the cytoadherent cloned isolate ItG2F6, and their phenotypes were examined with respect to cytoadherence, the expression of "knobs," and agglutination of infected erythrocytes with rabbit antiserum. The only chromosomal change detectable by two-dimensional PFGE among subclones that differ from wild type in each of these three characteristics is a deletion of approximately 100 kilobases at one end of chromosome 2. This deletion includes the gene coding for KAHRP and the subtelomeric repeat designated rep20.     

Isolation and characterization of the plasma membrane of human erythrocytes infected with the malarial parasite Plasmodium falciparum.

Human erythrocytes infected with the malarial parasite Plasmodium falciparum were labeled metabolically with a mixture of 15 radioactive amino acids. When synchronously growing parasites were at the schizont stage of development infected cells were concentrated and purified by using a Percoll-Hypaque gradient. The plasma membrane of the infected erythrocyte, isolated by binding cells to a solid support (Affi-Gel 731, Bio-Rad), was less than 1% contaminated with parasite membranes. Erythrocyte membrane proteins were analyzed by polyacrylamide gel electrophoresis and autoradiography. Despite the high sensitivity of the procedure, there was no evidence for the insertion of parasite proteins into the infected host cell membrane. One possible exception is a Mr 230,000 parasite protein present maximally as 9,000 copies per infected erythrocyte membrane. Moreover, no differences in the membrane proteins were observed between a highly knobby clone and a knobless clone of the same strain of P. falciparum. These findings appear to rule out the presence of parasite protein(s) playing a structural role in the formation of knobs on the erythrocyte surface and question whether the antigenic determinants on the P. falciparum-infected erythrocyte are of parasite origin or whether such antigens represent newly exposed or chemically modified erythrocyte determinants.     

Molecular basis for the dichotomy in Plasmodium falciparum adhesion to CD36 and chondroitin sulfate A

Plasmodium falciparum-infected erythrocytes adhere dichotomously to the host receptors CD36 and chondroitin sulfate A (CSA). This dichotomy is associated with parasite sequestration to microvasculature beds (CD36) or placenta (CSA), leading to site-specific pathogenesis. Both properties are mediated by members of the variant P. falciparum erythrocyte membrane protein 1 (PfEMP-1) family and reside on nonoverlapping domains of the molecule. To identify the molecular basis for the apparent dichotomy, we expressed various domains of PfEMP-1 individually or in combination and tested their binding properties. We found that the CD36-binding mode of the cysteine-rich interdomain region-1 (CIDR1) ablates the ability of the Duffy binding-like gamma domain to bind CSA. In contrast, neither a non-CD36-binding CIDR1 nor an intercellular adhesion molecule 1 binding domain had any affect on CSA binding. Our findings point out that interactions between different domains of PfEMP-1 can alter the adhesion phenotype of infected erythrocytes and provide a molecular basis for the apparent dichotomy in adhesion. We suggest that the basis for the dichotomy is structural and that mutually exclusive conformations of PfEMP-1 are involved in binding to CD36 or CSA. Furthermore, we propose a model explaining the requirement for structural dichotomy between placental and nonplacental isolates.     

Knob antigen deposition in cerebral malaria

Plasmodium falciparum-infected erythrocytes attach to the endothelial cells via electron-dense knobs and this attachment has been suggested as one of the contributing factors in the development of cerebral malaria. Monoclonal antibodies against an 80-95 Kd knob protein were prepared and applied to brain tissue from cerebral malaria patients. The deposition of the 80-95 Kd knob protein antibodies was observed in the basement membrane of cerebral capillaries by the peroxidase anti-peroxidase method. This result indicates involvement of knob protein deposition in the pathogenesis of cerebral malaria.     

Heparan sulfate on endothelial cells mediates the binding of Plasmodium falciparum-infected erythrocytes via the DBL1alpha domain of PfEMP1

Plasmodium falciparum may cause severe forms of malaria when excessive sequestration of infected and uninfected erythrocytes occurs in vital organs. The capacity of wild-type isolates of P falciparum-infected erythrocytes (parasitized red blood cells [pRBCs]) to bind glycosaminoglycans (GAGs) such as heparin has been identified as a marker for severe disease. Here we report that pRBCs of the parasite FCR3S1.2 and wild-type clinical isolates from Uganda adhere to heparan sulfate (HS) on endothelial cells. Binding to human umbilical vein endothelial cells (HUVECs) and to human lung endothelial cells (HLECs) was found to be inhibited by HS/heparin or enzymes that remove HS from cell surfaces. (35)S-labeled HS extracted from HUVECs bound directly to the pRBCs' membrane. Using recombinant proteins corresponding to the different domains of P falciparum erythrocyte membrane protein 1 (PfEMP1), we identified Duffy-binding-like domain-1alpha (DBL1alpha) as the ligand for HS. DBL1alpha bound in an HS-dependent way to endothelial cells and blocked the adherence of pRBCs in a dose-dependent manner. (35)S-labeled HS bound to DBL1alpha-columns and eluted as a distinct peak at 0.4 mM NaCl. (35)S-labeled chondroitin sulfate (CS) of HUVECs did not bind to PfEMP1 or to the pRBCs' membrane. Adhesion of pRBCs of FCR3S1.2 to platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, mediated by the cysteine-rich interdomain region 1alpha (CIDR1alpha), was found be operative with, but independent of, the binding to HS. HS and the previously identified HS-like GAG on uninfected erythrocytes may act as coreceptors in endothelial and erythrocyte binding of rosetting parasites, causing excessive sequestration of both pRBCs and RBCs.     

The surface variant antigens of Plasmodium falciparum contain cross-reactive epitopes

Plasmodium falciparum parasites evade the host immune system by clonal expression of the variant antigen, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Antibodies to PfEMP1 correlate with development of clinical immunity but are predominantly variant-specific. To overcome this major limitation for vaccine development, we set out to identify cross-reactive epitopes on the surface of parasitized erythrocytes (PEs). We prepared mAbs to the cysteine-rich interdomain region 1 (CIDR1) of PfEMP1 that is functionally conserved for binding to CD36. Two mAbs, targeting different regions of CIDR1, reacted with multiple P. falciparum strains expressing variant PfEMP1s. One of these mAbs, mAb 6A2-B1, recognized nine of 10 strains tested, failing to react with only one strain that does not bind CD36. Flow cytometry with Chinese hamster ovary cells expressing variant CIDR1s demonstrated that both mAbs recognized the CIDR1 of various CD36-binding PfEMP1s and are truly cross-reactive. The demonstration of cross-reactive epitopes on the PE surface provides further credence for development of effective vaccines against the variant antigen on the surface of P. falciparum-infected erythrocytes.     

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes.

Cerebral involvement in Plasmodium falciparum malaria is associated with sequestration of infected red blood cells and occlusion of cerebral vessels. Adhesion of infected erythrocytes along the vascular endothelium as well as binding of uninfected erythrocytes to cells infected with late-stage asexual parasites (rosetting) may be important in erythrocyte sequestration. We report that the recently discovered rosetting phenomenon shares characteristics with other human cell-cell interactions (heparin sensitivity, temperature independence, Ca2+/Mg2+ and pH dependence). Mono- and polyclonal antibodies specific for PfHRP1, a histidine-rich protein present in the membrane of P. falciparum-infected erythrocytes, disrupt rosettes but do not affect attachment of infected erythrocytes to endothelial cells. The inhibitory anti-PfHRP1 antibodies reacted with rosetting parasites in indirect immunofluorescence and with P. falciparum polypeptides of Mr 28,000 and Mr 90,000 in immunoprecipitation and immunoblotting, respectively. No inhibitory effects on erythrocyte rosetting were obtained with antibodies to related histidine-rich or other antigens of P. lophurae or P. falciparum. Whether the epitope that mediates rosetting, and is recognized by the anti-PfHRP1 antibodies, is located on PfHRP1 or on a crossreactive antigen remains to be established. The results suggest that endothelial cytoadherence and erythrocyte rosetting involve different molecular mechanisms.     

Characterization of a protein correlated with the production of knob-like protrusions on membranes of erythrocytes infected with Plasmodium falciparum

Membranes of erythrocytes infected with Plasmodium falciparum develop protrusions called "knobs." These protrusions are not apparent on erythrocytes infected with young parasites (rings) but develop with the growth of parasites to the trophozoite and schizont stages. The nature and origin of knobs were characterized by comparing the stage-specific proteins of two culture lines of P. falciparum, K+ and K-. K+ parasites produce knobs; K- parasites do not. Erythrocytes infected with both types of parasites were labeled metabolically and samples were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels. There were no apparent differences in Coomassie blue-stained or radioactive components of rings of K+ and K- parasites. However, erythrocytes infected with K+ trophozoites or schizonts showed a major labeled protein, with an apparent molecular weight of approximately 80,000, that was not present in any developmental stage of K- parasites or in K+ rings. A fraction enriched in membranes from erythrocytes infected with K+ trophozoites showed enrichment of this protein. The results indicate that this protein, synthesized by the parasites, is correlated with the formation of knobs on the host cell membrane. Two additional labeled components were identified. These appeared with the development of schizonts in both K+ and K- parasites and are therefore stage-dependent and not correlated with knobs.     

Plasmodium falciparum intercellular adhesion molecule-1-based cytoadherence-related signaling in human endothelial cells

BACKGROUND: Cytoadherence of Plasmodium falciparum-infected erythrocytes to host endothelium has been associated with pathology in severe malaria, but, despite extensive information on the primary processes involved in the adhesive interactions, the mechanisms underlying disease are poorly understood. METHODS: We compared parasite lines varying in their binding properties to human endothelial cells for their ability to stimulate signaling activity. RESULTS: In human umbilical vein endothelial cells (HUVECs), which rely on adhesion to intercellular adhesion molecule (ICAM)-1 for binding, signaling is related to the avidity of the parasite line for ICAM-1 and can be blocked either through the use of anti-ICAM-1 monoclonal antibodies or HUVECs with altered ICAM-1 binding properties (i.e., ICAM-1(Kilifi)). Human dermal microvascular endothelial cells (HDMECs), which can bind infected erythrocytes via ICAM-1 and CD36, have a more complex pattern of signaling behavior, but this is also dependent on adhesive interactions rather than merely contact between cells. CONCLUSIONS: Signaling via apposition of P. falciparum-infected erythrocytes with host endothelium is dependent, at least in part, on the cytoadherence characteristics of the invading isolate. An understanding of the postadhesive processes produced by cytoadherence may help us to understand the variable pathologies seen in malaria disease.     

Adhesive functions of platelets lacking glycoprotein IV (CD36).

Glycoprotein IV (GPIV; CD36 or GPIIIb) is a cell surface glycoprotein that has been proposed as mediating a number of physiologically important processes such as the adhesion of platelets to thrombospondin (TSP) and collagen, the cytoadherence of Plasmodium falciparum-infected erythrocytes, and the TSP-dependent interaction of monocytes with platelets and macrophages. Because platelets of the Naka-negative phenotype have recently been shown to lack detectable GPIV, their availability offered the opportunity to test directly these hypotheses regarding its adhesive functions. It has been found that Naka-negative platelets and monocytes do not support cytoadherence of P falciparum-infected erythrocytes. Naka-negative platelets are deficient in the initial stages of their adhesion to fibrillar collagen and this defect is most marked under Mg(2+)-free conditions. Finally, the ability of Naka-negative platelets to bind TSP before or after activation is unimpaired as compared with normal controls. These results do not support a role for GPIV as the TSP receptor.     

A role for CD36 in the regulation of dendritic cell function

Dendritic cells (DC) are crucial for the induction of immune responses and thus an inviting target for modulation by pathogens. We have previously shown that Plasmodium falciparum-infected erythrocytes inhibit the maturation of DCs. Intact P. falciparum-infected erythrocytes can bind directly to CD36 and indirectly to CD51. It is striking that these receptors, at least in part, also mediate the phagocytosis of apoptotic cells. Here we show that antibodies against CD36 or CD51, as well as exposure to early apoptotic cells, profoundly modulate DC maturation and function in response to inflammatory signals. Although modulated DCs still secrete tumor necrosis factor-alpha, they fail to activate T cells and now secrete IL-10. We therefore propose that intact P. falciparum-infected erythrocytes and apoptotic cells engage similar pathways regulating DC function. These findings may have important consequences for the treatment of malaria and may suggest strategies for modulating pathological immune responses in autoimmune diseases.     

Immunity to malarial antigens on the surface of Plasmodium falciparum-infected erythrocytes

An indirect immunofluorescence test with fresh non-fixed infected blood as antigen was used to show that antibody in human sera from the Gambia recognized antigens on the surface of Plasmodium falciparum-infected human erythrocytes. Surface immunofluorescence was detected on 90% of erythrocytes infected with trophozoites and schizonts produced in continuous culture of isolates from the Gambia (FCR 3/K+), Brazil and Thailand. Fluorescence was equally strong with a Gambian parasite clone (FCR 3/K-) that lacked knobs, an ultrastructural modification of the erythrocyte membrane associated with parasite sequestration. Immunofluorescence could not be detected with an isolate from Uganda. The surface antigenicity of parasitized erythrocytes was eliminated by chymotrypsin and trypsin treatment. Fluorescence was specific for the surface of trophozoite- and schizont-infected cells on the condition that fresh erythrocytes were added to cultures every 4-5 days (subculture); if fresh erythrocytes were not added for over 2 weeks, a large percentage of non-infected erythrocytes also bound antibody. Normal erythrocytes incubated with media from these cultures also gave positive surface immunofluorescence. Thus, there are two types of antigenicity on erythrocytes: one expressed on infected erythrocytes and another passively absorbed from media to normal erythrocytes when parasites are not subcultured for long periods.     

Human cerebral malaria

Possible factors contributing to the development of cerebral malaria were discussed based on pathological changes in Burmese patients who died of cerebral malaria. Blockage of cerebral capillaries by Plasmodium falciparum infected erythrocytes appeared to be the principal cause of cerebral malaria. From electron microscopic results, it was concluded that knobs on infected erythrocytes acted as focal junctions which mediated adhesion to endothelial cells. The knobs are, therefore, important contributors to the blockage of the capillary lumen and ensuing pathological changes in cerebral tissues. Host cell molecules such as OKM5 and thrombospondin may function as endothelial cell surface receptors for the attachment of knobs of P. falciparum infected erythrocytes. Immunological events might also play a role in the pathogenesis of cerebral malaria. This was suggested by the presence of IgG, IgM, P. falciparum antigens, and knob proteins in the cerebral capillaries of the people with cerebral malaria. It will be important to assess the candidate malaria vaccines now in development not only for their efficacy in reducing parasitemia but for effects they may have on the sequestration of infected erythrocytes in the brain.     

Variants of Plasmodium falciparum erythrocyte membrane protein 1 expressed by different placental parasites are closely related and adhere to chondroitin sulfate A

Plasmodium falciparum-infected erythrocytes adhere to syncytiotrophoblast cells lining the placenta via glycosaminoglycans, such as chondroitin sulfate A (CSA) and hyaluronic acid. Adherence of infected erythrocytes to host receptors is mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP-1). A single PfEMP-1 domain (duffy binding-like [DBL]-3, of the gamma sequence class) from laboratory-adapted strains is thought to be responsible for binding to CSA. In this study, DBL-gamma domains expressed by placental P. falciparum isolates were shown to have an affinity to CSA. All parasite populations accumulating in infected placentas express only 1 variant of PfEMP-1, each of which contains a DBL-gamma domain with CSA binding capacities. Furthermore, sequence analysis data provide evidence for antigenic conservation among the DBL-gamma sequences expressed by different placental parasites. This study offers a close reflection of the process of parasite adhesion in the placenta and is crucial to the understanding of the pathogenesis of malaria during pregnancy.