Complement receptor 1 is the human erythrocyte receptor for Plasmodium vivax erythrocyte binding protein.

The discovery that Africans were resistant to infection by Plasmodium vivax (P. vivax) led to the conclusion that P. vivax invasion relied on the P. vivax Duffy Binding Protein (PvDBP) interacting with the Duffy Antigen Receptor for Chemokines (DARC) expressed on erythrocytes. However, the recent reporting of P. vivax infections in DARC-negative Africans suggests that the parasite might use an alternate invasion pathway to infect DARC-negative reticulocytes. To identify the parasite ligands and erythrocyte receptors that enable P. vivax invasion of both DARC-positive and -negative erythrocytes, we expressed region II containing the Duffy Binding-Like (DBL) domain of P. vivax erythrocyte binding protein (PvEBP-RII) and verified that the DBL domain binds to both DARC-positive and -negative erythrocytes. Furthermore, an AVidity-based EXtracelluar Interaction Screening (AVEXIS) was used to identify the receptor for PvEBP among over 750 human cell surface receptor proteins, and this approach identified only Complement Receptor 1 (CR1, CD35, or C3b/C4b receptor) as a PvEBP receptor. CR1 is a well-known receptor for P. falciparum Reticulocyte binding protein Homology 4 (PfRh4) and is present on the surfaces of both reticulocytes and normocytes, but its expression decreases as erythrocytes age. Indeed, PvEBP-RII bound to a subpopulation of both reticulocytes and normocytes, and this binding was blocked by the addition of soluble CR1 recombinant protein, indicating that CR1 is the receptor of PvEBP. In addition, we found that the Long Homology Repeat A (LHR-A) subdomain of CR1 is the only subdomain responsible for mediating the interaction with PvEBP-RII.

Vivax malaria and Duffy antigen: Stop being so negative.

Duffy blood group antigen (Duffy antigen/receptor for chemokines, atypical chemokine receptor-1, Duffy antigen), an essential Plasmodium vivax invasion receptor, is absent in most Africans. In this issue, two papers show erythroid precursors from Duffy-negative individuals transiently surface-express Duffy antigen and support vivax infection, potentially explaining low-density vivax infections across Africa.

The direct binding of Plasmodium vivax AMA1 to erythrocytes defines a RON2-independent invasion pathway.

We used a transgenic parasite in which Plasmodium falciparum parasites were genetically modified to express Plasmodium vivax apical membrane antigen 1 (PvAMA1) protein in place of PfAMA1 to study PvAMA1-mediated invasion. In P. falciparum, AMA1 interaction with rhoptry neck protein 2 (RON2) is known to be crucial for invasion, and PfRON2 peptides (PfRON2p) blocked the invasion of PfAMA1 wild-type parasites. However, PfRON2p has no effect on the invasion of transgenic parasites expressing PvAMA1 indicating that PfRON2 had no role in the invasion of PvAMA1 transgenic parasites. Interestingly, PvRON2p blocked the invasion of PvAMA1 transgenic parasites in a dose-dependent manner. We found that recombinant PvAMA1 domains 1 and 2 (rPvAMA1) bound to reticulocytes and normocytes indicating that PvAMA1 directly interacts with erythrocytes during the invasion, and invasion blocking of PvRON2p may result from it interfering with PvAMA1 binding to erythrocytes. It was previously shown that the peptide containing Loop1a of PvAMA1 (PvAMA1 Loop1a) is also bound to reticulocytes. We found that the Loop1a peptide blocked the binding of PvAMA1 to erythrocytes. PvAMA1 Loop1a has no polymorphisms in contrast to other PvAMA1 loops and may be an attractive vaccine target. We thus present the evidence that PvAMA1 binds to erythrocytes in addition to interacting with PvRON2 suggesting that the P. vivax merozoites may exploit complex pathways during the invasion process.

Contrasting epidemiology and genetic variation of Plasmodium vivax infecting Duffy-negative individuals across Africa.

OBJECTIVES: Plasmodium vivax malaria was thought to be rare in Africans who lack the Duffy blood group antigen expression. However, recent studies indicate that P. vivax can infect Duffy-negative individuals and has spread into areas of high Duffy negativity across Africa. Our study compared epidemiological and genetic features of P. vivax between African regions. METHODS: A standardized approach was used to identify and quantify P. vivax from Botswana, Ethiopia, and Sudan, where Duffy-positive and Duffy-negative individuals coexist. The study involved sequencing the Duffy binding protein (DBP) gene and inferring genetic relationships among P. vivax populations across Africa. RESULTS: Among 1215 febrile patients, the proportions of Duffy negativity ranged from 20-36% in East Africa to 84% in southern Africa. Average P. vivax prevalence among Duffy-negative populations ranged from 9.2% in Sudan to 86% in Botswana. Parasite density in Duffy-negative infections was significantly lower than in Duffy-positive infections. P. vivax in Duffy-negative populations were not monophyletic, with P. vivax in Duffy-negative and Duffy-positive populations sharing similar DBP haplotypes and occurring in multiple, well-supported clades. CONCLUSIONS: Duffy-negative Africans are not resistant to P. vivax, and the public health significance of this should not be neglected. Our study highlights the need for a standardized approach and more resources/training directed towards the diagnosis of vivax malaria in Africa.

Plasmodium vivax Infections Detected in a Large Number of Febrile Duffy-Negative Africans in Dschang, Cameroon.

The Duffy blood group is a critical receptor for Plasmodium vivax (P. vivax) invasion of red blood cells, and consequently, P. vivax infections were considered rare in sub-Saharan Africa where the prevalence of Duffy-negativity is high. However, recently, P. vivax infections have been found in Duffy-negative Africans throughout the malaria transmission area of sub-Saharan Africa, raising important questions concerning the molecular composition of these P. vivax clones and the red blood cell receptors that facilitate their invasion. Here, we describe an unusually high number of P. vivax infections in febrile Duffy-negative Africans in Dschang, Cameroon (177 of 500 outpatients), as compared with Santchou (two of 400 outpatients) and Kyé-ossi (two of 101 outpatients), in other areas in Cameroon. In the discussion, we speculate on the possible reasons why Dschang might account for the unusually large numbers of P. vivax infections in Duffy-negative individuals living there.

Whole genome sequencing of Plasmodium vivax isolates reveals frequent sequence and structural polymorphisms in erythrocyte binding genes.

Plasmodium vivax malaria is much less common in Africa than the rest of the world because the parasite relies primarily on the Duffy antigen/chemokine receptor (DARC) to invade human erythrocytes, and the majority of Africans are Duffy negative. Recently, there has been a dramatic increase in the reporting of P. vivax cases in Africa, with a high number of them being in Duffy negative individuals, potentially indicating P. vivax has evolved an alternative invasion mechanism that can overcome Duffy negativity. Here, we analyzed single nucleotide polymorphism (SNP) and copy number variation (CNV) in Whole Genome Sequence (WGS) data from 44 P. vivax samples isolated from symptomatic malaria patients in southwestern Ethiopia, where both Duffy positive and Duffy negative individuals are found. A total of 123,711 SNPs were detected, of which 22.7% were nonsynonymous and 77.3% were synonymous mutations. The largest number of SNPs were detected on chromosomes 9 (24,007 SNPs; 19.4% of total) and 10 (16,852 SNPs, 13.6% of total). There were particularly high levels of polymorphism in erythrocyte binding gene candidates including merozoite surface protein 1 (MSP1) and merozoite surface protein 3 (MSP3.5, MSP3.85 and MSP3.9). Two genes, MAEBL and MSP3.8 related to immunogenicity and erythrocyte binding function were detected with significant signals of positive selection. Variation in gene copy number was also concentrated in genes involved in host-parasite interactions, including the expansion of the Duffy binding protein gene (PvDBP) on chromosome 6 and MSP3.11 on chromosome 10. Based on the phylogeny constructed from the whole genome sequences, the expansion of these genes was an independent process among the P. vivax lineages in Ethiopia. We further inferred transmission patterns of P. vivax infections among study sites and showed various levels of gene flow at a small geographical scale. The genomic features of P. vivax provided baseline data for future comparison with those in Duffy-negative individuals and allowed us to develop a panel of informative Single Nucleotide Polymorphic markers diagnostic at a micro-geographical scale.

Antigen Discovery, Bioinformatics and Biological Characterization of Novel Immunodominant Babesia microti Antigens.

Babesia microti is an intraerythrocytic parasite and the primary causative agent of human babesiosis. It is transmitted by Ixodes ticks, transfusion of blood and blood products, organ donation, and perinatally. Despite its global public health impact, limited progress has been made to identify and characterize immunodominant B. microti antigens for diagnostic and vaccine use. Using genome-wide immunoscreening, we identified 56 B. microti antigens, including some previously uncharacterized antigens. Thirty of the most immunodominant B. microti antigens were expressed as recombinant proteins in E. coli. Among these, the combined use of two novel antigens and one previously described antigen provided 96% sensitivity and 100% specificity in identifying B. microti antibody containing sera in an ELISA. Using extensive computational sequence and bioinformatics analyses and cellular localization studies, we have clarified the domain architectures, potential biological functions, and evolutionary relationships of the most immunodominant B. microti antigens. Notably, we found that the BMN-family antigens are not monophyletic as currently annotated, but rather can be categorized into two evolutionary unrelated groups of BMN proteins respectively defined by two structurally distinct classes of extracellular domains. Our studies have enhanced the repertoire of immunodominant B. microti antigens, and assigned potential biological function to these antigens, which can be evaluated to develop novel assays and candidate vaccines.

A Way Forward for Culturing Plasmodium vivax.

Trager and Jensen established a method for culturing Plasmodium falciparum, a breakthrough for malaria research worldwide. Since then, multiple attempts to establish Plasmodium vivax in continuous culture have failed. Unlike P. falciparum, which can invade all aged erythrocytes, P. vivax is restricted to reticulocytes. Thus, a constant supply of reticulocytes is considered critical for continuous P. vivax growth in vitro. A critical question remains why P. vivax selectively invades reticulocytes? What do reticulocytes offer to P. vivax that is not present in mature erythrocytes? One possibility is protection from oxidative stress by glucose-6-phosphate dehydrogenase (G6PD). Here, we also suggest supplements to the media and procedures that may reduce oxidative stress and, as a result, establish a system for the continuous culture of P. vivax.

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.

Frequent expansion of Plasmodium vivax Duffy Binding Protein in Ethiopia and its epidemiological significance.

Plasmodium vivax invasion of human erythrocytes depends on the Duffy Binding Protein (PvDBP) which interacts with the Duffy antigen. PvDBP copy number has been recently shown to vary between P. vivax isolates in Sub-Saharan Africa. However, the extent of PvDBP copy number variation, the type of PvDBP multiplications, as well as its significance across broad samples are still unclear. We determined the prevalence and type of PvDBP duplications, as well as PvDBP copy number variation among 178 Ethiopian P. vivax isolates using a PCR-based diagnostic method, a novel quantitative real-time PCR assay and whole genome sequencing. For the 145 symptomatic samples, PvDBP duplications were detected in 95 isolates, of which 81 had the Cambodian and 14 Malagasy-type PvDBP duplications. PvDBP varied from 1 to >4 copies. Isolates with multiple PvDBP copies were found to be higher in symptomatic than asymptomatic infections. For the 33 asymptomatic samples, PvDBP was detected with two copies in two of the isolates, and both were the Cambodian-type PvDBP duplication. PvDBP copy number in Duffy-negative heterozygotes was not significantly different from that in Duffy-positives, providing no support for the hypothesis that increased copy number is a specific association with Duffy-negativity, although the number of Duffy-negatives was small and further sampling is required to test this association thoroughly.

Transcriptome profiling of Plasmodium vivax in Saimiri monkeys identifies potential ligands for invasion.

Unlike the case in Asia and Latin America, Plasmodium vivax infections are rare in sub-Saharan Africa due to the absence of the Duffy blood group antigen (Duffy antigen), the only known erythrocyte receptor for the P. vivax merozoite invasion ligand, Duffy binding protein 1 (DBP1). However, P. vivax infections have been documented in Duffy-negative individuals throughout Africa, suggesting that P. vivax may use ligands other than DBP1 to invade Duffy-negative erythrocytes through other receptors. To identify potential P. vivax ligands, we compared parasite gene expression in Saimiri and Aotus monkey erythrocytes infected with P. vivax Salvador I (Sal I). DBP1 binds Aotus but does not bind to Saimiri erythrocytes; thus, P. vivax Sal I must invade Saimiri erythrocytes independent of DBP1. Comparing RNA sequencing (RNAseq) data for late-stage infections in Saimiri and Aotus erythrocytes when invasion ligands are expressed, we identified genes that belong to tryptophan-rich antigen and merozoite surface protein 3 (MSP3) families that were more abundantly expressed in Saimiri infections compared with Aotus infections. These genes may encode potential ligands responsible for P. vivax infections of Duffy-negative Africans.

Plasmodium vivax Infections of Duffy-Negative Erythrocytes: Historically Undetected or a Recent Adaptation?

Plasmodium vivax is the main cause of malarial disease in Asia and South America. Plasmodium vivax infection was thought to be absent in African populations who are Duffy blood group antigen negative (Duffy-negative). However, many cases of P. vivax infection have recently been observed in Duffy-negative Africans. This raises the question: were P. vivax infections in Duffy-negative populations previously missed or has P. vivax adapted to infect Duffy-negative populations? This review focuses on recent P. vivax findings in Africa and reports views on the parasite ligands that may play a role in Duffy-negative P. vivax infections. In addition, clues gained from studying P. vivax infection of reticulocytes are presented, which may provide possible avenues for establishing P. vivax culture in vitro.

PfCDPK1 is critical for malaria parasite gametogenesis and mosquito infection.

Efforts to knock out Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) from asexual erythrocytic stage have not been successful, indicating an indispensable role of the enzyme in asexual growth. We recently reported generation of a transgenic parasite with mutant CDPK1 [Bansal A, et al. (2016) MBio 7:e02011-16]. The mutant CDPK1 (T145M) had reduced activity of transphosphorylation. We reasoned that CDPK1 could be disrupted in the mutant parasites. Consistent with this assumption, CDPK1 was successfully disrupted in the mutant parasites using CRISPR/Cas9. We and others could not disrupt PfCDPK1 in the WT parasites. The CDPK1 KO parasites show a slow growth rate compared with the WT and the CDPK1 T145M parasites. Additionally, the CDPK1 KO parasites show a defect in both male and female gametogenesis and could not establish an infection in mosquitoes. Complementation of the KO parasite with full-length PfCDPK1 partially rescued the asexual growth defect and mosquito infection. Comparative global transcriptomics of WT and the CDPK1 KO schizonts using RNA-seq show significantly high transcript expression of gametocyte-specific genes in the CDPK1 KO parasites. This study conclusively demonstrates that CDPK1 is a good target for developing transmission-blocking drugs.

P. falciparum RH5-Basigin interaction induces changes in the cytoskeleton of the host RBC.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.

A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes.

UNLABELLED: Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71(high)) Duffy-positive (Fy(+)) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes. IMPORTANCE: For decades, P. vivax infections in humans have been defined by a unique requirement for the interaction between the Duffy binding protein ligand of the parasite and the Duffy blood group antigen receptor (DARC). Recent reports of P. vivax infections in Duffy-negative individuals challenge this paradigm and suggest an alternate pathway of infection, potentially using the recently discovered EBP2. However, we demonstrate that EBP2 host cell specificity is more restricted than DBP binding and that EBP2 binds preferentially to Duffy-positive, young reticulocytes. This finding indicates that this DBP paralog does mediate a Duffy-independent pathway of infection.

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.

Role of Plasmodium vivax Duffy-binding protein 1 in invasion of Duffy-null Africans.

The ability of the malaria parasite Plasmodium vivax to invade erythrocytes is dependent on the expression of the Duffy blood group antigen on erythrocytes. Consequently, Africans who are null for the Duffy antigen are not susceptible to P. vivax infections. Recently, P. vivax infections in Duffy-null Africans have been documented, raising the possibility that P. vivax, a virulent pathogen in other parts of the world, may expand malarial disease in Africa. P. vivax binds the Duffy blood group antigen through its Duffy-binding protein 1 (DBP1). To determine if mutations in DBP1 resulted in the ability of P. vivax to bind Duffy-null erythrocytes, we analyzed P. vivax parasites obtained from two Duffy-null individuals living in Ethiopia where Duffy-null and -positive Africans live side-by-side. We determined that, although the DBP1s from these parasites contained unique sequences, they failed to bind Duffy-null erythrocytes, indicating that mutations in DBP1 did not account for the ability of P. vivax to infect Duffy-null Africans. However, an unusual DNA expansion of DBP1 (three and eight copies) in the two Duffy-null P. vivax infections suggests that an expansion of DBP1 may have been selected to allow low-affinity binding to another receptor on Duffy-null erythrocytes. Indeed, we show that Salvador (Sal) I P. vivax infects Squirrel monkeys independently of DBP1 binding to Squirrel monkey erythrocytes. We conclude that P. vivax Sal I and perhaps P. vivax in Duffy-null patients may have adapted to use new ligand-receptor pairs for invasion.

STEVOR is a Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion and rosetting.

Variant surface antigens play an important role in Plasmodium falciparum malaria pathogenesis and in immune evasion by the parasite. Although most work to date has focused on P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), two other multigene families encoding STEVOR and RIFIN are expressed in invasive merozoites and on the infected erythrocyte surface. However, their role during parasite infection remains to be clarified. Here we report that STEVOR functions as an erythrocyte-binding protein that recognizes Glycophorin C (GPC) on the red blood cell (RBC) surface and that its binding correlates with the level of GPC on the RBC surface. STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation), while antibodies targeting STEVOR in the merozoite can effectively inhibit invasion. Our results suggest a PfEMP1-independent role for STEVOR in enabling infected erythrocytes at the schizont stage to form rosettes and in promoting merozoite invasion.

Small molecule targeting malaria merozoite surface protein-1 (MSP-1) prevents host invasion of divergent plasmodial species.

Malaria causes nearly 1 million deaths annually. Recent emergence of multidrug resistance highlights the need to develop novel therapeutic interventions against human malaria. Given the involvement of sugar binding plasmodial proteins in host invasion, we set out to identify such proteins as targets of small glycans. Combining multidisciplinary approaches, we report the discovery of a small molecule inhibitor, NIC, capable of inhibiting host invasion through interacting with a major invasion-related protein, merozoite surface protein-1 (MSP-1). This interaction was validated through computational, biochemical, and biophysical tools. Importantly, treatment with NIC prevented host invasion by Plasmodium falciparum and Plasmodium vivax--major causative organisms of human malaria. MSP-1, an indispensable antigen critical for invasion and suitably localized in abundance on the merozoite surface represents an ideal target for antimalarial development. The ability to target merozoite invasion proteins with specific small inhibitors opens up a new avenue to target this important pathogen.

Triggers of key calcium signals during erythrocyte invasion by Plasmodium falciparum.

Invasion of erythrocytes by Plasmodium falciparum merozoites is a complex multi-step process mediated by specific interactions between host receptors and parasite ligands. Reticulocyte-binding protein homologues (RHs) and erythrocyte-binding-like (EBL) proteins are discharged from specialized organelles and used in early steps of invasion. Here we show that monoclonal antibodies against PfRH1 (an RH) block merozoite invasion by specifically inhibiting calcium signalling in the parasite, whereas invasion-inhibiting monoclonal antibodies targeting EBA175 (an EBL protein) have no effect on signalling. We further show that inhibition of this calcium signalling prevents EBA175 discharge and thereby formation of the junction between parasite and host cell. Our results indicate that PfRH1 has an initial sensing as well as signal transduction role that leads to the subsequent release of EBA175. They also provide new insights on how RH-host cell interactions lead to essential downstream signalling events in the parasite, suggesting new targets for malaria intervention.