Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.

Using the PfEMP1 Head Structure Binding Motif to Deal a Blow at Severe Malaria

Plasmodium falciparum (Pf) malaria causes 200 million cases worldwide, 8 million being severe and complicated leading to similar to 1 million deaths and similar to 100,000 abortions annually. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) has been implicated in cytoadherence and infected erythrocyte rosette formation, associated with cerebral malaria; chondroitin sulphate-A attachment and infected erythrocyte sequestration related to pregnancy-associated malaria and other severe forms of disease. An endothelial cell high activity binding peptide is described in several of this similar to 300 kDa hypervariable protein's domains displaying a conserved motif (GACxPxRRxxLC); it established H-bonds with other binding peptides to mediate red blood cell group A and chondroitin sulphate attachment. This motif (when properly modified) induced PfEMP1-specific strain-transcending, fully-protective immunity for the first time in experimental challenge in Aotus monkeys, opening the way forward for a long sought-after vaccine against severe malaria.

An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.

"A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies"

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface

A sequence in subdomain 2 of DBL1 alpha of plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1 alpha previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1 alpha-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1 alpha antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface.

The South American Plasmodium falciparum var gene repertoire is limited, highly shared and possibly lacks several antigenic types

The Plasmodium falciparum var gene family encodes large variant antigens, which are important virulence factors, and also targets of the humoral host response. The frequently observed mild outcomes of falciparum malaria in many places of the Amazon area prompted us to ask whether a globally restricted variant (var) gene repertoire is present in currently circulating and older isolates of this area. By exhaustive analysis of var gene tags from 89 isolates and clones taken during many years from all over the Brazilian Amazon, we estimate that there are probably no more than 350-430 distinct sequence types, less than for any similar sized area studied so far. Detailed analysis of the var tags from genetically distinct clones obtained from single isolates revealed restricted and redundant repertoires suggesting either a low incidence of infective bites or restricted variant gene diversity in inoculated parasites. Additionally, we found a structuring of var gene repertoires observed as a higher pairwise typing sharing in isolates from the same microregion compared to isolates from different regions. Fine analysis of translated var tags revealed that certain Distinct Sequence Identifiers (DSIDs) were differently represented in Brazilian/South American isolates when compared to datasets from other continents. By global alignment of worldwide var DBL alpha sequences and sorting in groups with more than 76% identity, 125 clusters were formed and more than half of all genes were found in nine clusters with 50 or more sequences. While Brazilian/South American sequences were represented only in 64 groups, African sequences were found in the majority of clusters. DSID type 1 related sequences accumulated almost completely in one single cluster, indicating that limited recombination occurs in these specific var gene types. These data demonstrate the so far highest pairwise type sharing values for the var gene family in isolates from all over an entire subcontinent. The apparent lack of specific sequences types suggests that the P. falciparum transmission dynamics in the whole Amazon are probably different from any other endemic region studied and possibly interfere with the parasite`s ability to efficiently diversify its variant gene repertoires. (C) 2010 Elsevier B.V. All rights reserved.

Protein export in Plasmodium parasites : from the endoplasmic reticulum to the vacuolar export machine

It is somewhat paradoxical that the malaria parasite’s survival strategy involves spending almost all of its blood-stage existence residing behind a two-membrane barrier in a host red blood cell, yet giving considerable attention to exporting parasite-encoded proteins back across these membranes. These exported proteins are thought to play diverse roles and are crucial in pathogenic processes, such as re-modelling of the erythrocyte cytoskeleton and mediating the export of a major virulence protein known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), and in metabolic processes such as nutrient uptake and solute exchange. Despite these varied roles most exported proteins have at least one common link; they share a trafficking pathway that begins with entry into the endoplasmic reticulum and concludes with passage across the vacuole membrane via a proteinaceous translocon known as the Plasmodium translocon of exported proteins (PTEX). In this commentary we review recent advances in our understanding of this export pathway and suggest several models by which different aspects of the process may be interconnected.

PTEX is an essential nexus for protein export in malaria parasites

During the blood stages of malaria, several hundred parasite-encoded proteins are exported beyond the double-membrane barrier that separates the parasite from the host cell cytosol. These proteins have a variety of roles that are essential to virulence or parasite growth. There is keen interest in understanding how proteins are exported and whether common machineries are involved in trafficking the different classes of exported proteins. One potential trafficking machine is a protein complex known as the Plasmodium translocon of exported proteins (PTEX). Although PTEX has been linked to the export of one class of exported proteins, there has been no direct evidence for its role and scope in protein translocation. Here we show, through the generation of two parasite lines defective for essential PTEX components (HSP101 or PTEX150), and analysis of a line lacking the non-essential component TRX2 (ref. 12), greatly reduced trafficking of all classes of exported proteins beyond the double membrane barrier enveloping the parasite. This includes proteins containing the PEXEL motif (RxLxE/Q/D) and PEXEL-negative exported proteins (PNEPs). Moreover, the export of proteins destined for expression on the infected erythrocyte surface, including the major virulence factor PfEMP1 in Plasmodium falciparum, was significantly reduced in PTEX knockdown parasites. PTEX function was also essential for blood-stage growth, because even a modest knockdown of PTEX components had a strong effect on the parasite's capacity to complete the erythrocytic cycle both in vitro and in vivo. Hence, as the only known nexus for protein export in Plasmodium parasites, and an essential enzymic machine, PTEX is a prime drug target.

Identification of a Plasmodium falciparum intercellular adhesion molecule-1 binding domain: A parasite adhesion trait implicated in cerebral malaria

Binding of infected erythrocytes to brain venules is a central pathogenic event in the lethal malaria disease complication, cerebral malaria. The only parasite adhesion trait linked to cerebral sequestration is binding to intercellular adhesion molecule-1 (ICAM-1). In this report, we show that Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) binds ICAM-1. We have cloned and expressed PfEMP1 recombinant proteins from the A4tres parasite. Using heterologous expression in mammalian cells, the minimal ICAM-1 binding domain was a complex domain consisting of the second Duffy binding-like (DBL) domain and the C2 domain. Constructs that contained either domain alone did not bind ICAM-1. Based on phylogenetic criteria, there are five distinct PfEMP1 DBL types designated α, β, γ, δ, and ɛ. The DBL domain from the A4tres that binds ICAM-1 is DBLβ type. A PfEMP1 cloned from a distinct ICAM-1 binding variant, the A4 parasite, contains a DBLβ domain and a C2 domain in tandem arrangement similar to the A4tres PfEMP1. Anti-PfEMP1 antisera implicate the DBLβ domain from A4var PfEMP1 in ICAM-1 adhesion. The identification of a P. falciparum ICAM-1 binding domain may clarify mechanisms responsible for the pathogenesis of cerebral malaria and lead to interventions or vaccines that reduce malarial disease.

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.

Plasmodium falciparum erythrocyte membrane protein 1 is a parasitized erythrocyte receptor for adherence to CD36, thrombospondin, and intercellular adhesion molecule 1.

Adherence of mature Plasmodium falciparum parasitized erythrocytes (PRBCs) to microvascular endothelium contributes directly to acute malaria pathology. We affinity purified molecules from detergent extracts of surface-radioiodinated PRBCs using several endothelial cell receptors known to support PRBC adherence, including CD36, thrombospondin (TSP), and intercellular adhesion molecule 1 (ICAM-1). All three host receptors affinity purified P. falciparum erythrocyte membrane protein 1 (PfEMP1), a very large malarial protein expressed on the surface of adherent PRBCs. Binding of PfEMP1 to particular host cell receptors correlated with the binding phenotype of the PRBCs from which PfEMP1 was extracted. Preadsorption of PRBC extracts with anti-PfEMP1 antibodies, CD36, or TSP markedly reduced PfEMP1 binding to CD36 or TSP. Mild trypsinization of intact PRBCs of P. falciparum strains shown to express antigenically different PfEMP1 released different (125)I-labeled tryptic fragments of PfEMP1 that bound specifically to CD36 and TSP. In clone C5 and strain MC, these activities resided on different tryptic fragments, but a single tryptic fragment from clone ItG-ICAM bound to both CD36 and TSP. Hence, the CD36- and TSP-binding domains are distinct entities located on a single PfEMP1 molecule. PfEMP1, the malarial variant antigen on infected erythrocytes, is therefore a receptor for CD36, TSP, and ICAM-1. A therapeutic approach to block or reverse adherence of PRBCs to host cell receptors can now be pursued with the identification of PfEMP1 as a malarial receptor for PRBC adherence to host proteins.

Modeling the development of acquired clinical immunity to Plasmodium falciparum malaria

Individuals living in regions where malaria is endemic develop an acquired immunity to malaria which enables them to remain asymptomatic while still carrying parasites. Field studies indicate that cumulative exposure to a variety of diverse Plasmodium parasites is required for the transition from symptomatic to asymptomatic malaria. This study used a simulation model of the within-host dynamics of P. falciparum to investigate the development of acquired clinical immunity under different transmission conditions and levels of parasite diversity. Antibodies developed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), a clonally variant molecule, were assumed to be a key human immunological response to P. falciparum infection, along with responses to clonally conserved but polymorphic antigens. The time to the development of clinical immunity was found to be proportional to parasite diversity and inversely proportional to transmission intensity. The effect of early termination of symptomatic infections by chemotherapy was investigated and found not to inhibit the host's ability to develop acquired immunity. However, the time required to achieve this state was approximately double that compared to when no treatment was administered. This study demonstrates that an immune response primarily targeted against PfEMP1 has the ability to reduce clinical symptoms of infections irrespective of whether treatment is administered, supporting its role in the development of acquired clinical immunity. The results also illustrate a novel use for simulation models of P. falciparum infections, investigation of the influence of intervention strategies on the development of naturally acquired clinical immunity.