Invasion by P. falciparum merozoites suggests a hierarchy of molecular interactions

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.     

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

Invasion by the malaria merozoite depends on recognition of specific erythrocyte surface receptors by parasite ligands. Plasmodium falciparum uses multiple ligands, including at least two gene families, reticulocyte binding protein homologues (RBLs) and erythrocyte binding proteins/ligands (EBLs). The combination of different RBLs and EBLs expressed in a merozoite defines the invasion pathway utilized and could also play a role in parasite virulence. The binding regions of EBLs lie in a conserved cysteine-rich domain while the binding domain of RBL is still not well characterized. Here, we identify the erythrocyte binding region of the P. falciparum reticulocyte binding protein homologue 1 (PfRH1) and show that antibodies raised against the functional binding region efficiently inhibit invasion. In addition, we directly demonstrate that changes in the expression of RBLs can constitute an immune evasion mechanism of the malaria merozoite.     

Reticulocyte-binding protein homologue 5 - An essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum

Invasion of erythrocytes is a prerequisite in the life history of the malaria parasite. Members of the reticulocyte-binding homologue family (PfRh) have been implicated in the invasion process and in some cases have been shown to act as adhesins, binding to specific receptors on the erythrocyte surface. We have identified a further, putatively essential, PfRh family member in the most virulent human malaria Plasmodium falciparum, called PfRh5, which binds to an unknown class of glycosylated receptors on the erythrocyte surface. This protein is an atypical PfRh family member, being much smaller than others and lacking a transmembrane and cytosolic region at the C-terminus. This suggests it may be part of a functional protein complex. PfRh5 localises to the rhoptries in merozoites and follows the tight junction during the process of erythrocyte invasion. Furthermore, rabbit immune serum raised against a portion of the ecto-domain, inhibits parasite invasion in vitro. We hypothesise an essential role for the PfRh5 adhesin in erythrocyte selection and commitment to invasion. Given its small size, we believe PfRh5 may prove to be a valuable candidate for inclusion in a multi-component anti-malarial vaccine.     

Identification of Plasmodium falciparum RhopH3 protein peptides that specifically bind to erythrocytes and inhibit merozoite invasion

The identification of sequences involved in binding to erythrocytes is an important step for understanding the molecular basis of merozoite-erythrocyte interactions that take place during invasion of the Plasmodium falciparum malaria parasite into host cells. Several molecules located in the apical organelles (micronemes, rhoptry, dense granules) of the invasive-stage parasite are essential for erythrocyte recognition, invasion, and establishment of the nascent parasitophorous vacuole. Particularly, it has been demonstrated that rhoptry proteins play an important role in binding to erythrocyte surface receptors, among which is the PfRhopH3 protein, which triggers important immune responses in patients from endemic regions. It has also been reported that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes, further supporting its direct involvement in erythrocyte invasion processes. In this study, PfRhopH3 consecutive peptides were synthesized and tested in erythrocyte binding assays for identifying those regions mediating binding to erythrocytes. Fourteen PfRhopH3 peptides presenting high specific binding activity were found, whose bindings were saturable and presented nanomolar dissociation constants. These high-activity binding peptides (HABPs) were characterized by having alpha-helical structural elements, as determined by circular dichroism, and having receptors of a possible sialic acid-dependent and/or glycoprotein-dependent nature, as evidenced in enzyme-treated erythrocyte binding assays and further corroborated by cross-linking assay results. Furthermore, these HABPs inhibited merozoite in vitro invasion of normal erythrocytes at 200 microM by up to 60% and 90%, suggesting that some RhopH3 protein regions are involved in the P. falciparum erythrocyte invasion.     

Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion

Some human malaria Plasmodium falciparum parasites, but not others, also cause disease in Aotus monkeys. To identify the basis for this variation, we crossed two clones that differ in Aotus nancymaae virulence and mapped inherited traits of infectivity to erythrocyte invasion by linkage analysis. A major pathway of invasion was linked to polymorphisms in a putative erythrocyte binding protein, PfRH5, found in the apical region of merozoites. Polymorphisms of PfRH5 from the A. nancymaae-virulent parent transformed the nonvirulent parent to a virulent parasite. Conversely, replacements that removed these polymorphisms from PfRH5 converted a virulent progeny clone to a nonvirulent parasite. Further, a proteolytic fragment of PfRH5 from the infective parasites bound to A. nancymaae erythrocytes. Our results also suggest that PfRH5 is a parasite ligand for human infection, and that amino acid substitutions can cause its binding domain to recognize different human erythrocyte surface receptors.     

Proteome analysis reveals a large merozoite surface protein-1 associated complex on the Plasmodium falciparum merozoite surface

Plasmodium merozoite surface protein-1 (MSP-1) is an essential antigen for the merozoite invasion of erythrocytes. A key challenge to the development of an effective malaria vaccine that can block the erythrocyte invasion is to establish the molecular interaction(s) among the parasite surface proteins as well as with the host cell encoded receptors. In the present study, we applied molecular interactions and proteome approaches to identify PfMSP-1 associated complex on the merozoite surface. Proteomic analysis identified a major malaria surface protein, PfRhopH3 interacting with PfMSP-1(42). Pull-down experiments with merozoite lysate using anti-PfMSP-1 or anti-PfRhopH3 antibodies showed 16 bands that when identified by tandem mass spectrometry corresponded to11 parasite proteins: PfMSP-3, PfMSP-6, PfMSP-7, PfMSP-9, PfRhopH3, PfRhopH1, PfRAP-1, PfRAP-2, and two RAP domain containing proteins. This MSP-1 associated complex was specifically seen at schizont/merozoite stages but not the next ring stage. We could also identify many of these proteins in culture supernatant, suggesting the shedding of the complex. Interestingly, the PfRhopH3 protein also showed binding to the human erythrocyte and anti-PfRhopH3 antibodies blocked the erythrocyte invasion of the merozoites. These results have potential implications in the development of PfMSP-1 based blood stage malaria vaccine.     

A thrombospondin structural repeat containing rhoptry protein from Plasmodium falciparum mediates erythrocyte invasion

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin‐treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy forthe development of vaccines against blood stage malaria parasites.     

Reticulocyte-binding protein homologue 1 is required for sialic acid-dependent invasion into human erythrocytes by Plasmodium falciparum

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand-receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The DeltaPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand-receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.     

RBC Barcoding Allows for the Study of Erythrocyte Population Dynamics and P. falciparum Merozoite Invasion

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion.     

Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen.     

Malaria Parasite Signal Peptide Peptidase is an ER‐Resident Protease Required for Growth but not for Invasion

The establishment of parasite infection within the human erythrocyte is an essential stage in the development of malaria disease. As such, significant interest has focused on the mechanics that underpin invasion and on characterization of parasite molecules involved. Previous evidence has implicated a presenilin‐like signal peptide peptidase (SPP) from the most virulent human malaria parasite, Plasmodium falciparum, in the process of invasion where it has been proposed to function in the cleavage of the erythrocyte cytoskeletal protein Band 3. The role of a traditionally endoplasmic reticulum (ER) protease in the process of red blood cell invasion is unexpected. Here, using a combination of molecular, cellular and chemical approaches we provide evidence that PfSPP is, instead, a bona fide ER‐resident peptidase that remains intracellular throughout the invasion process. Furthermore, SPP‐specific drug inhibition has no effect on erythrocyte invasion whilst having low micromolar potency against intra‐erythrocytic development. Contrary to previous reports, these results show that PfSPP plays no role in erythrocyte invasion. Nonetheless, PfSPP clearly represents a potential chemotherapeutic target to block parasite growth, supporting ongoing efforts to develop antimalarial‐targeting protein maturation and trafficking during intra‐erythrocytic development.     

The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.     

Export of parasite proteins to the erythrocyte in Plasmodium falciparum-infected cells

The human malaria parasite Plasmodium falciparum invades erythrocytes and develops within a parasitophorous vacuole. It has been proposed that constitutive protein export from the intracellular parasite is mediated by two types of secretory vesicles. One is targeted to the parasite plasma membrane and the other to a domain where the plasma and vacuolar membranes of the parasite are fused into a single bilayer. This differential targeting of vesicles may be regulated by the developmental stage of the parasite. Regulated secretion through the apical organelles at or immediately after the invasion of a new red cell may allow protein insertion at the erythrocyte surface and mediate formation of the joint membrane domain of constitutive secretion.     

The surface of the Plasmodium falciparum-infected erythrocyte

In order to navigate its complex lifecycle, the malaria parasites must interactwith a range of host cells. Examples of this are the invasion of hepatocytes by sporozoites and erythrocyte invasion by merozoites. This requirement for cell recognition brings with it the need to display cognate ligands on the parasite surface, and therefore the capacity of the host to develop defences against the infection. Even at a stage where the intracellular nature of erythrocyte development would appear to offer an opportunity for the parasite to be immunologically "silent", parasite-derived proteins are found on the surface of the infected erythrocyte. This review will discuss the proteins found on or associated with the surface of the infected erythrocyte and the resulting phenotypes.     

Getting down to malarial nuts and bolts: the interaction between Plasmodium vivax merozoites and their host erythrocytes

Of the four Plasmodium species that routinely cause malaria in humans, Plasmodium falciparum is responsible for the majority of malaria mortality and consequently gets most of the headlines. Outside Africa, however, more malaria cases are caused by its distant cousin Plasmodium vivax, resulting in a daunting morbidity and economic burden for countries across Asia and the Americas. Plasmodium life cycles are complex, but the symptoms and pathology of malaria occur during the blood phase, when merozoites recognize and invade erythrocytes, initiating a developmental programme that culminates in lysis of the erythrocyte and release of multiple daughter merozoites. P. vivax merozoites are dependent on a single host cell receptor for erythrocyte invasion, the Duffy antigen receptor for chemokines, and humans that do not express this receptor on the surface of their erythrocytes are immune to P. vivax infection. This essential receptor-ligand interaction is addressed from both the host and parasite side in two papers in this issue of Molecular Microbiology, with important implications for plans to develop a P. vivax vaccine.     

Localization and function of a Plasmodium falciparum protein (PF3D7_1459400) during erythrocyte invasion

Nearly 60% of Plasmodium falciparum proteins are still uncharacterized and their functions are unknown. In this report, we carried out the functional characterization of a 45 kDa protein (PF3D7_1459400) and showed its potential as a target for blood stage malaria vaccine development. Analysis of protein subcellular localization, native protein expression profile, and erythrocyte invasion inhibition of both clinical and laboratory parasite strains by peptide antibodies suggest a functional role of PF3D7_1459400 protein during erythrocyte invasion. Also, immunoreactivity screens using synthetic peptides of the protein showed that adults resident in malaria endemic regions in Ghana have naturally acquired plasma antibodies against PF3D7_1459400 protein. Altogether, this study presents PF3D7_1459400 protein as a potential target for the development of peptide-based vaccine for blood-stage malaria.Impact statementPlasmodium falciparum malaria is a global health problem. Erythrocyte invasion by P. falciparum merozoites appears to be a promising target to curb malaria. We have identified and characterized a novel protein that is involved in erythrocyte invasion. Our data on protein subcellular localization, stage-specific protein expression pattern, and merozoite invasion inhibition by α-peptide antibodies suggest a role for PF3D7_1459400 protein during P. falciparum erythrocyte invasion. Even more, the human immunoepidemiology data present PF3D7_1459400 protein as an immunogenic antigen which could be further exploited for the development of new anti-infective therapy against malaria.     

Deletion of a malaria invasion gene reduces death and anemia, in model hosts

Malaria parasites induce complex cellular and clinical phenotypes, including anemia, cerebral malaria and death in a wide range of mammalian hosts. Host genes and parasite 'toxins' have been implicated in malarial disease, but the contribution of parasite genes remains to be fully defined. Here we assess disease in BALB/c mice and Wistar rats infected by the rodent malaria parasite Plasmodium berghei with a gene knock out for merozoite surface protein (MSP) 7. MSP7 is not essential for infection but in P. falciparum, it enhances erythrocyte invasion by 20%. In vivo, as compared to wild type, the P. berghei Δmsp7 mutant is associated with an abrogation of death and a decrease from 3% to 2% in peak, circulating parasitemia. The Δmsp7 mutant is also associated with less anemia and modest increase in the size of follicles in the spleen. Together these data show that deletion of a single parasite invasion ligand modulates blood stage disease, as measured by death and anemia. This work is the first to assess the contribution of a gene present in all plasmodial species in severe disease.     

Molecular interactions and signaling mechanisms during erythrocyte invasion by malaria parasites

Invasion of erythrocytes by Plasmodium merozoites is a complex process that is mediated by specific molecular interactions. Here, we review recent studies on interactions between erythrocyte binding antigens (EBA) and PfRH proteins from the parasite and erythrocyte receptors involved in invasion. The timely release of these parasite ligands from internal organelles such as micronemes and rhoptries to the merozoite surface is critical for receptor-engagement leading to successful invasion. We review information on signaling mechanisms that control the regulated secretion of parasite proteins during invasion. Erythrocyte invasion involves the formation and movement of a junction between the invading merozoite and host erythrocyte. We review recent studies on the molecular composition of the junction and the molecular motor that drives movement of the junction.     

Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro

Michel R., Schupp K., Raether W. and Bierther F.W. 1980. Formation of a close junction during invasion of erythrocytes by Toxoplasma gondii in vitro. International Journal for Parasitology10: 309–313. During the investigation of erythrocyte entry by Toxoplasma trophozoites the formation of a close junction between the erythrocyte and the invading parasite at the site of contact could be observed. The erythrocyte membrane and the outer membrane of the parasite pellicle show increased electron density in the circumferential attachment zone at the orifice of the erythrocyte invagination. The erythrocyte membrane has a thickness in this area of 7–8.5 nm. This membrane differentiation can be observed at the early stages of invasion when the tip of the apical end of the parasite induces a small erythrocyte invagination.It is still preserved in further advanced stages, when the parasite enters the enlarging invagination of the erythrocyte. As the morphological features resemble those observed during merozoite invasion into erythrocytes we consider our findings may have a similar significance for the mechanism of invasion by Toxoplasma as for the erythrocyte entry by malaria parasites.     

Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.