Localization of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs

Plasmodium falciparum parasites that induce knobs in the host erythrocyte membrane (K+ phenotype) synthesize a 90 kDa histidine-rich protein (PfHRP-1), whereas knobless variants do not. A monoclonal antibody (mAb 89) to PfHRP-1, in combination with cryo-thin section immunoelectron microscopy, localized the antigen in the parasitophorous vacuolar space and vesicles within the erythrocyte cytosol. Additional immunoelectron microscopic studies showed that PfHRP-1 was also associated with submembranous electron-dense material under knobs and with microfilaments of the host erythrocyte skeletal network. Immunofluorescence and immunoelectron microscopy of intact, non-fixed K+ infected erythrocytes using mAb 89 and a rabbit antiserum raised against purified PfHRP-1, failed to identify any surface exposed epitopes. These antibodies also failed to block cytoadherence of infected erythrocytes to C32 melanoma cells or to affect macrophage phagocytosis of infected erythrocytes.     

Structure and expression of the knob-associated histidine-rich protein of Plasmodium falciparum

cDNA clones encoding 473 amino acids of the knob-associated histidine-rich protein (PfHRPI) of Plasmodium falciparum clone FCR-3/A2 (Gambia) have been isolated and sequenced. Although a short region close to the amino terminus of the predicted sequence contains three blocks of six, seven or nine consecutive histidine residues, the most abundant amino acid is lysine. The predicted sequence contains a putative amino-terminal signal sequence and two potential asparagine glycosylation sites. A 1284 bp Sau3A cDNA fragment was expressed in Escherichia coli as a fusion protein that was recognized by an anti-PfHRPI monoclonal antibody. Pulsed field gradient electrophoresis indicated that the PfHRPI gene is located on chromosome 2. The PfHRPI gene was present, apparently intact, in knobless parasites derived from one uncloned P. falciparum isolate (St. Lucia). In a knobless derivative of another uncloned isolate (Malayan Camp) and in a cloned knobless line (FCR-3/D4) of a third isolate (Gambian), that part of the gene covered by the cDNA clone has been deleted. Loss of PfHRPI expression may therefore arise via several different mechanisms of gene alteration.     

MESA is a Plasmodium falciparum phosphoprotein associated with the erythrocyte membrane skeleton

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum is an antigenically variable, high molecular weight protein of trophozoites and schizonts that is located at the erythrocyte surface membrane. It is first synthesized at the late ring stage and continues to be synthesized until late schizogony. MESA cannot be detected on the external surface of erythrocytes infected by trophozoites and early schizonts but is located at the internal surface in association with the erythrocyte membrane skeleton. The degree of association with the membrane skeleton varies among parasite lines, being greater in knobby parasite lines. MESA is phosphorylated and is present in a similar location to another phosphoprotein, the ring-infected erythrocyte surface antigen (RESA). However, it differs from RESA in being detected at a later stage of asexual development of the parasite.     

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.     

Classification of adhesive domains in the Plasmodium falciparum erythrocyte membrane protein 1 family

The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family of cytoadherent proteins has a central role in disease from malaria infection. This highly diverse gene family is involved in binding interactions between infected erythrocytes and host cells and is expressed in a clonally variant pattern at the erythrocyte surface. We describe by sequence analysis the structure and domain organization of 20 PfEMP1 from the GenBank database. Four domains comprise the majority of PfEMP1 extracellular sequence: the N-terminal segment (NTS) located at the amino terminus of all PfEMP1, the C2, the Cysteine-rich Interdomain Region (CIDR) and the Duffy Binding-like (DBL) domains. Previous work has shown that CIDR and DBL domains can possess adhesive properties. CIDR domains grouped as three distinct sequence classes (, β, and γ) and DBL domains as five sequence classes (, β, γ, δ, and ). Consensus motifs are described for the different DBL and CIDR types. Whereas the number of DBL and CIDR domains vary between PfEMP1, PfEMP1 domain architecture is not random in that certain tandem domain associations — such as DBLCIDR, DBLδCIDRβ, and DBLβC2 — are preferentially observed. This conservation may have functional significance for PfEMP1 folding, transport, or binding activity. Parasite binding phenotype appears to be a determinant of infected erythrocyte tissue tropism that contributes to parasite survival, transmission, and disease outcome. The sequence classification of DBL and CIDR types may have predictive value for identifying PfEMP1 domains with a particular binding property. This information might be used to develop interventions targeting parasite binding variants that cause disease.     

Characterization of the antibody response against Plasmodium falciparum erythrocyte membrane protein 1 in human volunteers

The immune response against the Plasmodium falciparum variant surface antigen P. falciparum erythrocyte membrane protein 1 (PfEMP1) is a key component of clinical immunity against falciparum malaria. In this study, we used sera from human volunteers who had been infected with the P. falciparum 3D7 strain to investigate the development, specificity, and dynamics of anti-PfEMP1 antibodies measured against six different strain 3D7 Duffy binding-like domain 1α (DBL1α) fusion proteins. We observed that a parasitemia of 20 to 200 infected erythrocytes per μl was required to trigger an antibody response to DBL1α and that antibodies against one DBL1α variant cross-react with other DBL1α variants. Both serum and purified immunoglobulin Gs (IgGs) were able to agglutinate infected erythrocytes, and purified anti-DBL1α IgGs bound to the live infected red blood cell surface in a punctate surface pattern, confirming that the IgGs recognize native PfEMP1. Analysis of sera from tourists naturally infected with P. falciparum suggests that the anti-PfEMP1 antibodies often persisted for more than 100 days after a single infection. These results help to further our understanding of the development of acquired immunity to P. falciparum infections.     

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.     

Immunoglobulin G antibody reactivity to a group A Plasmodium falciparum erythrocyte membrane protein 1 and protection from P. falciparum malaria

Variant surface antigens (VSA) on the surface of Plasmodium falciparum-infected red blood cells play a major role in the pathogenesis of malaria and are key targets for acquired immunity. The best-characterized VSA belong to the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. In areas where P. falciparum is endemic, parasites causing severe malaria and malaria in young children with limited immunity tend to express semiconserved PfEMP1 molecules encoded by group A var genes. Here we investigated antibody responses of Tanzanians who were 0 to 19 years old to PF11_0008, a group A PfEMP1. PF11_0008 has previously been found to be highly transcribed in a nonimmune Dutch volunteer experimentally infected with NF54 parasites. A high proportion of the Tanzanian donors had antibodies against recombinant PF11_0008 domains, and in children who were 4 to 9 years old the presence of antibodies to the PF11_0008 CIDR2beta domain was associated with reduced numbers of malaria episodes. These results indicate that homologues of PF11_0008 are present in P. falciparum field isolates and suggest that PF11_0008 CIDR2beta-reactive antibodies might be involved in protection against malaria episodes.     

Reactivity of a monoclonal antibody produced to the histidine-rich protein of Plasmodium lophurae with Plasmodium falciparum

Monoclonal antibodies were produced against the histidine-rich protein of Plasmodium lophurae and tested for reactivity with Plasmodium falciparum antigens. One anti-histidine-rich protein monoclonal antibody showed immunological cross-reactivity with polypeptides of P. falciparum synthesized in vivo and in vitro.     

Inhibition of dendritic cell maturation by malaria is dose dependent and does not require Plasmodium falciparum erythrocyte membrane protein 1

Red blood cells infected with Plasmodium falciparum (iRBCs) have been shown to modulate maturation of human monocyte-derived dendritic cells (DCs), interfering with their ability to activate T cells. Interaction between Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and CD36 expressed by DCs is the proposed mechanism, but we show here that DC modulation does not require CD36 binding, PfEMP1, or contact between DCs and infected RBCs and depends on the iRBC dose. iRBCs expressing a PfEMP1 variant that binds chondroitin sulfate A (CSA) but not CD36 were phagocytosed, inhibited lipopolysaccharide (LPS)-induced phenotypic maturation and cytokine secretion, and abrogated the ability of DCs to stimulate allogeneic T-cell proliferation. CD36- and CSA-binding iRBCs showed comparable inhibition. P. falciparum lines rendered deficient in PfEMP1 expression by targeted gene knockout or knockdown also inhibited LPS-induced phenotypic maturation, and separation of DCs and iRBCs in transwells showed that inhibition was not contact dependent. Inhibition was observed at an iRBC:DC ratio of 100:1 but not at a ratio of 10:1. High doses of iRBCs were associated with apoptosis of DCs, which was not activation induced. Lower doses of iRBCs stimulated DC maturation sufficient to activate autologous T-cell proliferation. In conclusion, modulation of DC maturation by P. falciparum is dose dependent and does not require interaction between PfEMP1 and CD36. Inhibition and apoptosis of DCs by high-dose iRBCs may or may not be physiological. However, our observation that low-dose iRBCs initiate functional DC maturation warrants reevaluation and further investigation of DC interactions with blood-stage P. falciparum.     

Genetic polymorphism at the C-terminal domain (region III) of knob-associated histidine-rich protein (KAHRP) of Plasmodium falciparum in isolates from Iran

The knob-associated histidine-rich protein (KAHRP) plays a major role in the virulence of Plasmodium falciparum and is one of the targets for molecular therapy. The primary structure of KAHRP of P. falciparum consists of three domains (regions I–III), of which the C-terminal domain (region III) is the most polymorphic segment of this protein. One of the main obstacles is genetic diversity in designing and developing of malaria control strategies such as molecular therapy and vaccines. The primary objective of the present study was to investigate and analyze the extent of genetic polymorphism at the region III of KAHRP of P. falciparum in isolates from Iran. A fragment of the kahrp gene spanning the C-terminal domain was amplified by nested PCR from 50 P. falciparum isolates collected from two malaria endemic areas of Iran during 2009 to August 2010 and sequenced. In this study, three allelic types were observed at the C-terminal domain of KAHRP on the basis of the molecular weight of nested PCR products and the obtained sequencing data. The presence of multiple alleles of the kahrp gene indicates that several P. falciparum strains exist in the malaria endemic areas of Iran. Our findings will be valuable in the design and the development of the molecular therapeutic reagents for falciparum malaria.     

Limited cross-reactivity among domains of the Plasmodium falciparum clone 3D7 erythrocyte membrane protein 1 family

The var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of P. falciparum clone 3D7 into groups A and B/A (category A) and groups B, B/C, and C (category non-A). Expression of category A molecules is associated with severe malaria, and that of category non-A molecules is associated with uncomplicated malaria and asymptomatic infection. Here we assessed cross-reactivity among 60 different recombinant PfEMP1 domains derived from clone 3D7 by using a competition enzyme-linked immunosorbent assay and a pool of plasma from 63 malaria-exposed Tanzanian individuals. We conclude that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains sharing cross-reactive antibody epitopes. Identification of groups of serological cross-reacting molecules is pivotal for the development of vaccines based on PfEMP1.     

The ring-infected erythrocyte surface antigen protein of Plasmodium falciparum is phosphorylated upon association with the host cell membrane

The ring-infected erythrocyte surface antigen (RESA) is a 155-kDa malarial polypeptide which is released from merozoites and becomes associated with the erythrocyte membrane at the time of invasion. Inside-out vesicles (IOVs) prepared from Plasmodium falciparum-infected erythrocytes contain RESA, presumably bound to the membrane skeleton, as it is largely insoluble in Triton X-100. When these IOVs were incubated with [gamma-32P]ATP, a 155-kDa polypeptide was labeled in IOVs from infected, but not from uninfected erythrocytes. Immunoprecipitation using specific rabbit antisera confirmed that RESA is indeed a phosphoprotein. Phosphoamino acid analysis revealed phosphoserine and a small amount of phosphothreonine, but no phosphotyrosine. Labeling of intact parasitized erythrocytes with inorganic [32P]phosphate for several hours in culture resulted in RESA in Triton-insoluble extracts being phosphorylated. Labeling of synchronized parasites showed that RESA was phosphorylated only when it became associated with the erythrocyte membrane, and although RESA was abundant in mature parasites, it was not phosphorylated. RESA, released into the culture supernatants during the growth of P. falciparum, bound to IOVs prepared from normal uninfected erythrocytes, and subsequent labeling with [gamma-32P]ATP resulted in the phosphorylation of RESA. The evidence suggests that RESA is phosphorylated by an erythrocyte membrane kinase and probably not by a parasite-encoded enzyme.     

A 60-kDa Plasmodium falciparum protein at the moving junction formed between merozoite and erythrocyte during invasion

Invasion of erythrocytes by malaria merozoites requires the formation of a junction of attachment between erythrocyte and merozoite membranes. The attachment junction initially forms at the apical region of the merozoite. It then moves around to the posterior of the merozoite as invasion proceeds. A monoclonal antibody against a 60-kDa merozoite protein (termed MCP-1 for merozoite capping protein 1) of Plasmodium falciparum reacts in an immunofluorescence pattern resembling the moving junction. By two-color immunofluorescence, MCP-1 was located at the attachment site formed between the merozoite apical region and erythrocyte. During invasion, MCP-1 separated and migrated around merozoites at the orifice of the parasitophorous vacuole. In newly-invaded erythrocytes, MCP-1 persisted at the pole of the young parasite nearest the erythrocyte membrane, suggesting its anterior-to-posterior movement. MCP-1 exhibited no variability in molecular mass among the FCR-3, Camp and 7G8 strains of P. falciparum, and the epitope was invariant in the P. falciparum strains studied. We conclude that MCP-1 may participate in merozoite invasion of erythrocytes by facilitating attachment or movement of the junction along the parasite cytoskeletal network.     

Expression of the histidine-rich protein PfHRP1 by knob-positive Plasmodium falciparum is not sufficient for cytoadherence of infected erythrocytes.

Six culture-adapted knob-positive Plasmodium falciparum parasites, four of which were nonbinding in an in vitro cytoadherence assay, were tested for the presence of the knob-associated histidine-rich protein PfHRP1. Two knob-positive, strongly cytoadherence-positive P. falciparum strains from Aotus monkeys were also examined. All parasites expressed PfHRP1, suggesting that it plays a structural or functional role related to knobs but is not by itself sufficient for cytoadherence of P. falciparum-infected erythrocytes.     

LANCL1, an erythrocyte protein recruited to the Maurer's clefts during Plasmodium falciparum development

As the malarial parasite Plasmodium falciparum develops inside the erythrocyte, parasite-derived membrane structures, referred to as Maurer's clefts, play an important role in parasite development by delivering parasite proteins to the host cell surface, and participating in the assembly of the cytoadherence complex, essential for the pathogenesis of cerebral malaria. PfSBP1 is an integral membrane protein of the clefts, interacting with an erythrocyte cytosolic protein, identified here as the human Lantibiotic synthetase component C-like protein LANCL1. LANCL1 is specifically recruited to the surface of Maurer's clefts in P. falciparum mature blood stages. We propose that the interaction between PfSBP1 and LANCL1 is central for late steps of the parasite development to prevent premature rupture of the red blood cell membrane.