Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth.     

Role of calcineurin and actin dynamics in regulated secretion of microneme proteins in Plasmodium falciparum merozoites during erythrocyte invasion

Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane‐bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K⁺) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca²⁺), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca²⁺ are not completely understood. Here, we show that a P. falciparum Ca²⁺‐dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca²⁺‐dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K⁺ environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K⁺ environment resulted in inhibition of secretion of microneme protein apical merozoite antigen‐1 (PfAMA‐1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be criticalfor microneme secretion. PfCN thus serves as an effector of Ca²⁺‐dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood‐stage parasites providing a novel approach towards development of new therapeutic strategies against malaria.     

Secreted protein with altered thrombospondin repeat (SPATR) is essential for asexual blood stages but not required for hepatocyte invasion by the malaria parasite Plasmodium berghei

Upon entering its mammalian host, the malaria parasite productively invades two distinct cell types, that is, hepatocytes and erythrocytes during which several adhesins/invasins are thought to be involved. Many surface-located proteins containing thrombospondin Type I repeat (TSR) which help establish host–parasite molecular crosstalk have been shown to be essential for mammalian infection. Previous reports indicated that antibodies produced against Plasmodium falciparum secreted protein with altered thrombospondin repeat (SPATR) block hepatocyte invasion by sporozoites but no genetic evidence of its contribution to invasion has been reported. After failing to generate Spatr knockout in Plasmodium berghei blood stages, a conditional mutagenesis system was employed. Here, we show that SPATR plays an essential role during parasite's blood stages. Mutant salivary gland sporozoites exhibit normal motility, hepatocyte invasion, liver stage development and rupture of the parasitophorous vacuole membrane resulting in merosome formation. But these mutant hepatic merozoites failed to establish a blood stage infection in vivo. We provide direct evidence that SPATR is not required for hepatocyte invasion but plays an essential role during the blood stages of P. berghei.     

PfRH2b specific monoclonal antibodies inhibit merozoite invasion

Erythrocyte invasion by merozoite is a multistep process involving multiple ligand–receptor interactions. The Plasmodium falciparum reticulocyte binding protein homologues (PfRHs) consists of five functional members. The differential expression of PfRHs has been linked to the utilization of different invasion pathways by the merozoites as well as a mechanism of immune evasion. PfRHs are expressed at the apical end of merozoite and form interactions with distinct red blood cell (RBC) surface receptors that are important for successful invasion. Here we show that PfRH2b undergoes processing before and during merozoite invasion. The different processed fragments bind to chymotrypsin sensitive RBC surface receptors. We also show that PfRH2b follows the merozoite tight junction during invasion. Monoclonal antibodies (mAbs) inhibit merozoites invasion by blocking tight junction formation. mAbs binding to PfRH2b block merozoites intracellular Ca²⁺ signal necessary for EBA175 surface expression. The data suggests that a conserved function of PfRHs, where their interaction with RBC surface receptors facilitated recruitment of EBA175 and other tight junction proteins necessary for merozoite invasion by modulating merozoite intracellular Ca²⁺ signals.     

The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K⁺ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca²⁺ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca²⁺ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.     

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes.     

Characterization of Plasmodium falciparum Calcium-dependent Protein Kinase 1 (PfCDPK1) and Its Role in Microneme Secretion during Erythrocyte Invasion

Calcium-dependent protein kinases (CDPKs) play important roles in the life cycle of Plasmodium falciparum and other apicomplexan parasites. CDPKs commonly have an N-terminal kinase domain (KD) and a C-terminal calmodulin-like domain (CamLD) with calcium-binding EF hands. The KD and CamLD are separated by a junction domain (JD). Previous studies on Plasmodium and Toxoplasma CDPKs suggest a role for the JD and CamLD in the regulation of kinase activity. Here, we provide direct evidence for the binding of the CamLD with the P3 region (Leu³⁵⁶ to Thr³⁷⁰) of the JD in the presence of calcium (Ca²⁺). Moreover, site-directed mutagenesis of conserved hydrophobic residues in the JD (F363A/I364A, L356A, and F350A) abrogates functional activity of PfCDPK1, demonstrating the importance of these residues in PfCDPK1 function. Modeling studies suggest that these residues play a role in interaction of the CamLD with the JD. The P3 peptide, which specifically inhibits the functional activity of PfCDPK1, blocks microneme discharge and erythrocyte invasion by P. falciparum merozoites. Purfalcamine, a previously identified specific inhibitor of PfCDPK1, also inhibits microneme discharge and erythrocyte invasion, confirming a role for PfCDPK1 in this process. These studies validate PfCDPK1 as a target for drug development and demonstrate that interfering with its mechanistic regulation may provide a novel approach to design-specific PfCDPK1 inhibitors that limit blood stage parasite growth and clear malaria parasite infections.     

A processing product of the Plasmodium falciparum reticulocyte binding protein RH1 shows a close association with AMA1 during junction formation

Plasmodium falciparum responsible for the most virulent form of malaria invades human erythrocytes through multiple ligand‐receptor interactions. The P. falciparum reticulocyte binding protein homologues (PfRHs) are expressed at the apical end of merozoites and form interactions with distinct erythrocyte surface receptors that are important for invasion. Here using a range of monoclonal antibodies (mAbs) against different regions of PfRH1 we have investigated the role of PfRH processing during merozoite invasion. We show that PfRH1 gets differentially processed during merozoite maturation and invasion and provide evidence that the different PfRH1 processing products have distinct functions during invasion. Using in‐situ Proximity Ligation and FRET assays that allow probing of interactions at the nanometre level we show that a subset of PfRH1 products form close association with micronemal proteins Apical Membrane Antigen 1 (AMA1) in the moving junction suggesting a critical role in facilitating junction formation and active invasion. Our data provides evidence that time dependent processing of PfRH proteins is a mechanism by which the parasite is able to regulate distinct functional activities of these large processes. The identification of a specific close association with AMA1 in the junction now may also provide new avenues to target these interactions to prevent merozoite invasion.     

A novel ligand from Plasmodium falciparum that binds to a sialic acid-containing receptor on the surface of human erythrocytes

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor-ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid-dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte-binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid-dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease-resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid-dependent receptor on human erythrocytes.     

Identifying novel Plasmodium falciparum erythrocyte invasion receptors using systematic extracellular protein interaction screens

The invasion of host erythrocytes by the parasite Plasmodium falciparum initiates the blood stage of infection responsible for the symptoms of malaria. Invasion involves extracellular protein interactions between host erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. Despite significant research effort, many merozoite surface ligands have no known erythrocyte binding partner, most likely due to the intractable biochemical nature of membrane‐tethered receptor proteins and their interactions. The few receptor–ligand pairs that have been described have largely relied on sourcing erythrocytes from patients with rare blood groups, a serendipitous approach that is unsatisfactory for systematically identifying novel receptors. We have recently developed a scalable assay called AVEXIS (for AVidity‐based EXtracellular Interaction Screen), designed to circumvent the technical difficulties associated with the identification of extracellular protein interactions, and applied it to identify erythrocyte receptors for orphan P. falciparum merozoite ligands. Using this approach, we have recently identified Basigin (CD147) and Semaphorin‐7A (CD108) as receptors for RH5 and MTRAP respectively. In this essay, we review techniques used to identify Plasmodium receptors and discuss how they could beapplied in the future to identify novel receptors both for Plasmodium parasites but also other pathogens.     

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.     

Human erythrocyte band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3–MSP1 complex

Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by P. falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the P. falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP1₁₉ and the 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1.     

A novel erythrocyte binding antigen-175 paralogue from Plasmodium falciparum defines a new trypsin-resistant receptor on human erythrocytes

The recognition and invasion of human erythrocytes by the most lethal malaria parasite Plasmodium falciparum is dependent on multiple ligand-receptor interactions. Members of the erythrocyte binding-like (ebl) family, including the erythrocyte binding antigen-175 (EBA-175), are responsible for high affinity binding to glycoproteins on the surface of the erythrocyte. Here we describe a paralogue of EBA-175 and show that this protein (EBA-181/JESEBL) binds in a sialic acid-dependent manner to erythrocytes. EBA-181 is expressed at the same time as EBA-175 and co-localizes with this protein in the microneme organelles of asexual stage parasites. The receptor binding specificity of EBA-181 to erythrocytes differs from other members of the ebl family and is trypsin-resistant and chymotrypsin-sensitive. Furthermore, using glycophorin B-deficient erythrocytes we show that binding of EBA-181 is not dependent on this sialoglycoprotein. The level of expression of EBA-181 differs among parasite lines, and the importance of this ligand for invasion appears to be strain-dependent as the EBA-181 gene can be disrupted in W2mef parasites, without affecting the invasion phenotype, but cannot be targeted in 3D7 parasites.     

A Bacterial Phosphatase-Like Enzyme of the Malaria Parasite Plasmodium falciparum Possesses Tyrosine Phosphatase Activity and Is Implicated in the Regulation of Band 3 Dynamics during Parasite Invasion

Eukaryotic parasites of the genus Plasmodium cause malaria by invading and developing within host erythrocytes. Here, we demonstrate that PfShelph2, a gene product of Plasmodium falciparum that belongs to the Shewanella-like phosphatase (Shelph) subfamily, selectively hydrolyzes phosphotyrosine, as shown for other previously studied Shelph family members. In the extracellular merozoite stage, PfShelph2 localizes to vesicles that appear to be distinct from those of rhoptry, dense granule, or microneme organelles. During invasion, PfShelph2 is released from these vesicles and exported to the host erythrocyte. In vitro, PfShelph2 shows tyrosine phosphatase activity against the host erythrocyte protein Band 3, which is the most abundant tyrosine-phosphorylated species of the erythrocyte. During P. falciparum invasion, Band 3 undergoes dynamic and rapid clearance from the invasion junction within 1 to 2 s of parasite attachment to the erythrocyte. Release of Pfshelph2 occurs after clearance of Band 3 from the parasite-host cell interface and when the parasite is nearly or completely enclosed in the nascent vacuole. We propose a model in which the phosphatase modifies Band 3 in time to restore its interaction with the cytoskeleton and thus reestablishes the erythrocyte cytoskeletal network at the end of the invasion process.     

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.     

Semaphorin-7A Is an Erythrocyte Receptor for P. falciparum Merozoite-Specific TRAP Homolog,MTRAP

The motility and invasion of Plasmodium parasites is believed to require a cytoplasmic actin-myosin motor associated with a cell surface ligand belonging to the TRAP (thrombospondin-related anonymous protein) family. Current models of invasion usually invoke the existence of specific receptors for the TRAP-family ligands on the surface of the host cell; however, the identities of these receptors remain largely unknown. Here, we identify the GPI-linked protein Semaphorin-7A (CD108) as an erythrocyte receptor for the P. falciparum merozoite-specific TRAP homolog (MTRAP) by using a systematic screening approach designed to detect extracellular protein interactions. The specificity of the interaction was demonstrated by showing that binding was saturable and by quantifying the equilibrium and kinetic biophysical binding parameters using surface plasmon resonance. We found that two MTRAP monomers interact via their tandem TSR domains with the Sema domains of a Semaphorin-7A homodimer. Known naturally-occurring polymorphisms in Semaphorin-7A did not quantitatively affect MTRAP binding nor did the presence of glycans on the receptor. Attempts to block the interaction during in vitro erythrocyte invasion assays using recombinant proteins and antibodies showed no significant inhibitory effect, suggesting the inaccessibility of the complex to proteinaceous blocking agents. These findings now provide important experimental evidence to support the model that parasite TRAP-family ligands interact with specific host receptors during cellular invasion.     

Genetic analysis of the cytoplasmic domain of the PfRh2b merozoite invasion protein of Plasmodium falciparum

Apicomplexan parasites employ multiple adhesive ligands for recognition and entry into host cells. The Duffy binding-like (DBL) and the reticulocyte binding protein-like (RBL) families are central to the invasion of erythrocytes by the malaria parasite. These type-1 transmembrane proteins are composed of large ectodomains and small conserved cytoplasmic tail domains. The cytoplasmic tail domain of the micronemal DBL protein EBA-175 is required for a functional ligand-receptor interaction, but not for correct trafficking and localisation. Here we focus on the cytoplasmic tail domain of the rhoptry-localised Plasmodium falciparum RBL PfRh2b. We have identified a conserved sequence of six amino acids, enriched in acidic residues, in the cytoplasmic tail domains of RBL proteins from Plasmodium spp. Genetic analyses reveal that the entire cytoplasmic tail and the conserved motif within the cytoplasmic tail are indispensable for invasion P. falciparum. Site-directed mutagenesis of the conserved moiety reveals that changes in the order of the amino acids of the conserved moiety, but not the charge of the sequence, can be tolerated. Shuffling of the motif has no effect on either invasion phenotype or PfRh2b expression and trafficking. Although the PfRh2b gene can be readily disrupted, our results suggest that modification of the PfRh2b cytoplasmic tail results in strong dominant negative activity, highlighting important differences between the PfRh2b and EBA-175 invasion ligands.     

Enhancing Blockade of Plasmodium falciparum Erythrocyte Invasion: Assessing Combinations of Antibodies against PfRH5 and Other Merozoite Antigens

No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC₅₀ values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen 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.     

The upstream sequence segment of the C-terminal cysteine-rich domain is required for microneme trafficking of Plasmodium falciparum erythrocyte binding antigen 175

Erythrocyte invasion is a critical step for survival of Plasmodium parasites, the causative agents of malaria, in their host and recognition of the host cell receptors by Plasmodium erythrocyte-binding-like (EBL) proteins plays an important role. Although EBL subcellular localization was shown to be closely linked to parasite virulence in the rodent model of malaria, the trafficking of EBL to micronemes, the secretory organelle in the invasive parasite is not fully understood. In this study, we assessed the impact of the deletion and amino acid replacement of Plasmodium falciparum EBL (EBA-175) using transgenic P. falciparum lines expressing modified EBA-175. We found that, in addition to a signal peptide and a cysteine rich region (region 6) to the cytoplasmic tail, a previously unrecognized sequence segment in region 5 was required for correct microneme trafficking of EBA-175. Replacement of Arg or Phe residues in this segment altered microneme trafficking, suggesting that the sequence itself contained critical information. Based on these findings, we propose that the sequence segment in region 5 is also required for the recognition of EBA-175 by the trafficking machinery to direct this protein to the microneme. Our results provide key information to clarify an as yet unidentified EBA-175 trafficking mechanism.