Epigenetic dysregulation of virulence gene expression in severe Plasmodium falciparum malaria

Chronic infections with the human malaria parasite Plasmodium falciparum depend on antigenic variation. P. falciparum erythrocyte membrane protein 1 (PfEMP1), the major erythrocyte surface antigen mediating parasite sequestration in the microvasculature, is encoded in parasites by a highly diverse family of var genes. Antigenic switching is mediated by clonal variation in var expression, and recent in vitro studies have demonstrated a role for epigenetic processes in var regulation. Expression of particular PfEMP1 variants may result in parasite enrichment in different tissues, a factor in the development of severe disease. Here, we study in vivo human infections and provide evidence that infection-induced stress responses in the host can modify PfEMP1 expression via the perturbation of epigenetic mechanisms. Our work suggests that severe disease may not be the direct result of an adaptive virulence strategy to maximize parasite survival but that it may indicate a loss of control of the carefully regulated process of antigenic switching that maintains chronic infections.     

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.     

Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria.     

Skeleton-binding protein 1 functions at the parasitophorous vacuole membrane to traffic PfEMP1 to the Plasmodium falciparum-infected erythrocyte surface

A key feature of Plasmodium falciparum, the parasite causing the most severe form of malaria in humans, is its ability to export parasite molecules onto the surface of the erythrocyte. The major virulence factor and variant surface protein PfEMP1 (P falciparum erythrocyte membrane protein 1) acts as a ligand to adhere to endothelial receptors avoiding splenic clearance. Because the erythrocyte is devoid of protein transport machinery, the parasite provides infrastructure for trafficking across membranes it traverses. In this study, we show that the P falciparum skeleton-binding protein 1 (PfSBP1) is required for transport of PfEMP1 to the P falciparum-infected erythrocyte surface. We present evidence that PfSBP1 functions at the parasitophorous vacuole membrane to load PfEMP1 into Maurer clefts during formation of these structures. Furthermore, the major reactivity of antibodies from malaria-exposed multigravid women is directed toward PfEMP1 because this is abolished in the absence of PfSBP1.     

Adhesive receptors on malaria-parasitized red cells

Antigenic variation, rosetting and cytoadhesion are key determinants in the survival and virulence of the malaria parasite Plasmodium falciparum. These properties reside in a multigene protein family called P. falciparum erythrocyte membrane protein I (PfEMPI), encoded by the large and diverse var gene family. PfEMPI plays a central role in the biology of P. falciparum and its interaction with the human host. The molecular mechanism and the domains involved in cytoadherence, rosetting and antigenic variation are beginning to unfold. Domains mediating rosetting and adhesion to several key host receptors have already been identified. Understanding the role of PfEMPI in the pathogenesis and survival of malaria parasites is the key for the development of anti-adhesion vaccines and therapeutics to reduce the mortality and morbidity of P. falciparum infections.     

Assignment of functional roles to parasite proteins in malaria-infected red blood cells by competitive flow-based adhesion assay

Adhesion of parasitized red blood cells (PRBCs) to endothelial cells and subsequent accumulation in the microvasculature are pivotal events in the pathogenesis of falciparum malaria. During intraerythrocytic development, numerous proteins exported from the parasite associate with the RBC membrane skeleton but the precise function of many of these proteins remain unknown. Their cellular location, however, suggests that some may play a role in adhesion. The adhesive properties of PRBCs are best studied under flow conditions in vitro; however, experimental variation in levels of cytoadherence in currently available assays make subtle alterations in adhesion difficult to quantify. Here, we describe a flow-based assay that can quantify small differences in adhesion and document the extent to which a number of parasite proteins influence adhesion using parasite lines that no longer express specific proteins. Loss of parasite proteins ring-infected erythrocyte surface antigen (RESA), knob-associated histidine-rich protein (KAHRP) or Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) had a significant effect on the ability of PRBCs to adhere, whereas loss of mature parasite-infected erythrocyte surface antigen (MESA) had no effect. Our studies indicate that a number of membrane skeleton-associated parasite proteins, although not exposed on the RBC surface, can collectively affect the adhesive properties of PRBCs and further our understanding of pathophysiologically relevant structure/function relationships in malaria-infected RBCs.     

Antigenic differences and conservation among placental Plasmodium falciparum-infected erythrocytes and acquisition of variant-specific and cross-reactive antibodies

Background. Pregnant women are infected by Plasmodium falciparum with novel antigenic phenotypes that adhere to chondroitin sulfate A (CSA) and other receptors in the placenta. The diverse and variant parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is encoded by var genes, is a ligand for CSA and a major target of antibodies associated with protective immunity.Methods. Serum samples from pregnant women exposed to malaria were tested for immunoglobulin G, adhesion-inhibitory antibodies, and agglutinating antibodies to different CSA-binding isolates expressing conserved var2csa-type genes and to parasite isolates from infected placentas. Parasite isolates also were examined to assess PfEMP1 expression, the effect of trypsin treatment of infected erythrocytes on parasite adhesion and cleavage of PfEMP1, and inhibition of adhesion by rabbit antiserum raised against a CSA-binding isolate.Results. Findings demonstrated that (1) there are significant antigenic differences between CSA-binding isolates that correspond with polymorphisms in var2csa; (2) there are differences in the properties of PfEMP1 and antibody reactivity between CSA-binding and placental isolates, which express multiple PfEMP1 forms; (3) acquired antibodies target diverse and cross-reactive epitopes expressed by CSA-binding infected erythrocytes, and cross-reactive antibodies are not necessarily cross-inhibitory; and (4) the breadth of antibody reactivity is greater among multigravidae than among primigravidae.Conclusions. Immunity may be mediated by a repertoire of antibodies to diverse and common epitopes. Strategies based on vaccination with a single domain or isolate might be hindered by antigenic diversity.     

恶性疟原虫表面抗原及其与致病的关系

恶性疟原虫在感染红细胞后,会将一些蛋白质运输到红细胞表面,这些蛋白质与红细胞本身的膜蛋白质相互作用,造成宿主细胞在形态学、生理学和功能上的本质变化,而由这些变化所导致的病理特征,正是每年150万～300万恶性疟患者死亡的主要原因之一.随着基因组学和生物化学的发展,在过去的十几年里,对于这些表面抗原分子的了解越来越深入.该文就恶性疟原虫起到黏附和抗原变异作用的分子进行了概述.     

Antigenic diversity amongst ten geographic isolates of Plasmodium falciparum defined by merozoite invasion inhibition assay.

The extent to which human antibodies involved in functional immunity react with antigenic determinants varying between different isolates or strains of human malaria parasite Plasmodium falciparum will influence the design of vaccine against malaria. In this study, in vitro inhibition of merozoite invasion in erythrocytes by an immune human serum was used to define the antigenic differences in 10 isolates of P. falciparum from three endemic areas, i.e. Africa, South America and Southeast Asia. The serum inhibited the invasion of merozoites of all the strains but the extent of inhibition varied from low to moderate to high degree indicating antigenic differences amongst isolates of P. falciparum. The antigenic differences could not be correlated to the geographic origin of the parasite isolate.     

Conserved residues in the Plasmodium vivax Duffy-binding protein ligand domain are critical for erythrocyte receptor recognition

Malaria merozoite invasion of human erythrocytes depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium vivax merozoite invasion is totally dependent on the recognition of the Duffy blood group antigen by the parasite ligand Duffy-binding protein (DBP). Receptor recognition by P. vivax relies on a cysteine-rich domain, the DBL domain or region II, at the N terminus of the extracellular portion of DBP. The minimal region of the DBP implicated for receptor recognition lies between cysteines 4 and 8 of the DBL domain, which is a region that also has the highest rate of allelic polymorphisms among parasite isolates. We previously found that allelic polymorphisms in this region altered the P. vivax DBL domain antigenic character, which contrasts with changes in receptor specificity attributed to polymorphisms in some homologous ligands of Plasmodium falciparum. To further investigate the relative importance of conserved and polymorphic residues within this DBL central region, we identified residues critical for receptor recognition by site-directed mutagenesis. Seventy-seven surface-predicted residues of the Sal-1 DBL domain were substituted with alanine and assayed for erythrocyte binding activity by expression of the mutant proteins on the surface of transiently transfected COS cells. The functional effect of alanine substitution varied from nil to complete loss of DBL erythrocyte-binding activity. Mutations that caused loss of ligand function mostly occurred in discontinuous clusters of conserved residues, whereas nearly all mutations in polymorphic residues did not affect erythrocyte binding. These data delineate DBL domain residues essential for receptor recognition.     

Rosetting: a new cytoadherence property of malaria-infected erythrocytes

Plasmodium fragile infection of the toque monkey is a natural host-parasite association in which parasite sequestration occurs as during P. falciparum infection of humans. We have studied parasite sequestration of P. fragile and demonstrated the existence of a new property of cytoadherence of infected erythrocytes, "rosetting," which is defined as the agglutination of uninfected erythrocytes around parasitized erythrocytes. Rosetting in vitro and sequestration in vivo appear simultaneously as the parasite matures. The spleen plays a role in modulating cytoadherence; both sequestration and rosetting, which occur with cloned parasites from spleen-intact animals, are markedly reduced in splenectomized animals infected with parasites derived from the same clone. Sequestration and rosetting can be reversed by immune serum. Protease treatment of infected blood abolishes rosetting; however, if treatment is performed at an early stage of schizogony, rosetting reappears if parasites are allowed to further develop in the absence of protease. These results indicate that with P. fragile in its natural primate host, rosetting and sequestration are related to the presence on the infected erythrocyte surface of a parasite-derived antigenic component, the expression of which is modulated by the spleen.     

Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte

In blood-stage infection by the human malaria parasite Plasmodium falciparum, export of proteins from the intracellular parasite to the erythrocyte is key to virulence. This export is mediated by a host-targeting (HT) signal present on a "secretome" of hundreds of parasite proteins engaged in remodeling the erythrocyte. However, the route of HT-mediated export is poorly understood. Here we show that minimal soluble and membrane protein reporters that contain the HT motif and mimic export of endogenous P falciparum proteins are detected in the lumen of "cleft" structures synthesized by the pathogen. Clefts are efficiently targeted by the HT signal. Furthermore, the HT signal does not directly translocate across the parasitophorous vacuolar membrane (PVM) surrounding the parasite to deliver protein to the erythrocyte cytoplasm, as suggested by current models of parasite protein trafficking to the erythrocyte. Rather, it is a lumenal signal that sorts protein into clefts, which then are exported beyond the PVM. These data suggest that Maurer's clefts, which are unique to the virulent P falciparum species, are pathogen-induced secretory organelles that concentrate HT-containing soluble and membrane parasite proteins in their lumen for delivery to the host erythrocyte.     

Virulence and transmission success of the malarial parasite Plasmodium falciparum

Virulence of Plasmodium falciparum is associated with the expression of variant surface antigens designated PfEMP1 (P. falciparum erythrocyte membrane protein 1) that are encoded by a family of var genes. Data presented show that the transmission stages of P. falciparum also express PfEMP1 variants. Virulence in this host-parasite system can be considered a variable outcome of optimizing the production of sexual transmission stages from the population of disease-inducing asexual stages. Immunity to PfEMP1 will contribute to the regulation of this trade-off by controlling the parasite population with potential to produce mature transmission stages.     

Plasmodium falciparum induces reorganization of host membrane proteins during intraerythrocytic growth

The virulence of the malaria parasite, Plasmodium falciparum, is due in large part to the way in which it modifies the membrane of its erythrocyte host. In this work we have used confocal microscopy and fluorescence recovery after photo-bleaching to examine the lateral mobility of host membrane proteins in erythrocytes infected with P falciparum at different stages of parasite growth. The erythrocyte membrane proteins band 3 and glycophorin show a marked decrease in mobility during the trophozoite stage of growth. Erythrocytes infected with a parasite strain that does not express the knob-associated histidine-rich protein show similar effects, indicating that this parasite protein does not contribute to the immobilization of the host proteins. Erythrocytes infected with ring-stage parasites exhibit intermediate mobility indicating that the parasite is able to modify its host prior to its active feeding stage.     

Adhesion of normal and Plasmodium falciparum ring-infected erythrocytes to endothelial cells and the placenta involves the rhoptry-derived ring surface protein-2

Recent findings have challenged the current view of Plasmodium falciparum (P falciparum) blood-stage biology by demonstrating the cytoadhesion of early ring-stage-infected erythrocytes (rIEs) to host endothelial cells and placental syncytiotrophoblasts. The adhesion of rIEs was observed only in parasites that bind to the placenta via chondroitin sulfate A (CSA). In this work, a panel of mouse monoclonal antibodies (mAbs) that specifically inhibit cytoadhesion of rIEs but not of mature IEs was generated The previously described ring surface protein 2 (RSP-2), a 42-kDa protein, was identified as the target of the ring-stage-specific mAbs. Time course surface fluorescence experiments revealed a short overlap (approximately 4 hours) of expression between RSP-2 and P falciparum erythrocyte membrane protein 1 (PfEMP1). Their consecutive expression enables IEs to adhere to endothelial cells during the entire blood-stage cycle. During this study, a new phenotype was detected in parasite cultures, the adhesion of normal erythrocytes (nEs) to endothelial cells. All adherent nEs were coated with RSP-2. Immunolocalization studies show that RSP-2 is a rhoptry-derived protein that is discharged onto the erythrocyte membrane during contact with merozoites. Our results identify RSP-2 as a key molecule in sequestration of young blood-stage forms and nEs to endothelial cells.