Falciparum Invasion Ligands Research Articles

Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion

AbstractThe malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Recent increase in low complexity polygenomic infections and sialic acid-independent invasion pathways in Plasmodium falciparum from Western Gambia

BackgroundThe malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles.MethodsBlood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates.ResultsThough the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years.ConclusionsOverall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.Graphical

Acquisition, maintenance and adaptation of invasion inhibitory antibodies against Plasmodium falciparum invasion ligands involved in immune evasion.

Erythrocyte-binding antigens (EBAs) and P. falciparum reticulocyte-binding homologue proteins (PfRhs) are two important protein families that can vary in expression and utilization by P. falciparum to evade inhibitory antibodies. We evaluated antibodies at repeated time-points among individuals living in an endemic region in Nigeria over almost one year against these vaccine candidates. Antibody levels against EBA140, EBA175, EBA181, PfRh2, PfRh4, and MSP2, were measured by ELISA. We also used parasites with disrupted EBA140, EBA175 and EBA181 genes to show that all these were targets of invasion inhibitory antibodies. However, antigenic targets of inhibitory antibodies were not stable and changed substantially over time in most individuals, independent of age. Antibodies levels measured by ELISA also varied within and between individuals over time and the antibodies against EBA181, PfRh2 and MSP2 declined more rapidly in younger individuals (≤15 years) compared with older (>15). The breadth of high antibody responses over time was more influenced by age than by the frequency of infection. High antibody levels were associated with a more stable invasion inhibitory response, which could indicate that during the long process of formation of immunity, many changes not only in levels but also in functional responses are needed. This is an important finding in understanding natural immunity against malaria, which is essential for making an efficacious vaccine.

Different Regions of Plasmodium falciparum Erythrocyte-Binding Antigen 175 Induce Antibody Responses to Infection of Varied Efficacy.

Increasing evidence suggests that antibodies against merozoite proteins involved in Plasmodium falciparum invasion into the red blood cell play an important role in clinical immunity to malaria. Erythrocyte-binding antigen 175 (EBA-175) is the best-characterized P. falciparum invasion ligand, reported to recognize glycophorin A on the surface of erythrocytes. Its protein structure comprises 6 extracellular regions. Whereas region II contains Duffy binding-like domains involved in the binding to glycophorin A, the functional role of regions III-V is less clear. We developed a novel cytometric bead array for assessment of antigen-specific antibody concentration in plasma to evaluate the efficacy of immune responses to different regions of EBA-175 and associations between antibody levels with protection from symptomatic malaria in a treatment-reinfection cohort study. We found that while antibodies to region II are highly abundant, circulating levels as low as 5-10 µg/mL of antibodies specific for region III or the highly conserved regions IV-V predict strong protection from clinical malaria. These results lend support for the development of conserved regions of EBA-175 as components in a combination of a malaria vaccine.

Analysis of Erythrocyte Invasion Mechanisms of Plasmodium falciparum Clinical Isolates Across 3 Malaria-Endemic Areas in Ghana.

Plasmodium falciparum invades human erythrocytes by using an array of ligands that interact with several receptors, including sialic acid (SA), complement receptor 1 (CR1), and basigin. We hypothesized that in malaria-endemic areas, parasites vary invasion pathways under immune pressure. Therefore, invasion mechanisms of clinical isolates collected from 3 zones of Ghana with different levels of endemicity (from lowest to highest, Accra, Navrongo, and Kintampo) were compared using standardized methods. Blood samples were collected from children aged 2-14 years in whom malaria was diagnosed, and erythrocyte invasion phenotypes were determined using the enzymes neuraminidase, chymotrypsin, and trypsin, which differentially cleave receptors from the erythrocyte surface. In addition, antibodies against CR1 and basigin were used to determine the contributions of these receptors to invasion. Gene expression levels of P. falciparum invasion ligands were also examined. The parasites generally expressed SA-independent invasion phenotypes across the malaria-endemic areas, with parasites from Kintampo showing the highest invasion rates in neuraminidase-treated erythrocytes. CR1 was a major mediator of SA-independent invasion, while basigin was essential for both SA-dependent and SA-independent invasion mechanisms. Furthermore, expression of the basigin ligand PfRh5 was the best predictor of donor parasitemia. Erythrocyte invasion phenotypes expressed by P. falciparum are influenced by endemicity levels, and the PfRh5-basigin pathway is a potential vaccine target.

Quantification of Plasmodium-host protein interactions on intact, unmodified erythrocytes by back-scattering interferometry.

BackgroundInvasion of host erythrocytes by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion involves recognition events between erythrocyte receptors and ligands on the merozoite, the invasive blood form of the parasite. Identifying and characterizing host-parasite interactions is impeded by the biochemical challenges of working with membrane-embedded glycoprotein receptors. For example, the interaction between P. falciparum erythrocyte binding antigen 175 (PfEBA175) and glycophorin A (GYPA) depends on post-translational modifications that are not easily added in recombinant expression systems, and the use of native GYPA is limited by the hydrophobic transmembrane region, making it difficult to biochemically manipulate. It would, therefore, be desirable to perform quantitative binding assays with receptors embedded within the membranes of intact human erythrocytes.MethodsThe extracellular region of GYPA was over-expressed as a soluble protein in HEK293E cells. This protein was characterized, sialylated and evaluated for binding to the PfEBA175 protein. The label-free and free-solution assay, backscattering interferometry (BSI), was used to perform binding assays of two well-characterized P. falciparum invasion ligands to intact unmodified human erythrocytes.ResultsFindings indicate that the post-translational modifications present on native GYPA are required for it to bind recombinant PfEBA175 and that these modifications cannot be recapitulated in vitro using mammalian overexpression methods. Here, BSI was used to obtain quantitative, high fidelity interaction determinations on intact, unmodified erythrocytes. Using BSI and purified recombinant proteins constituting the entire ectodomains of the P. falciparum merozoite ligands PfEBA175 and PfRH5, KDs of 1.1 μM and 50 nM were measured for the PfRH5-BSG and PfEBA175-GYPA interactions, respectively, in good agreement with previous biophysical measurements of these interactions.ConclusionsThese results demonstrate that BSI can be used to detect and quantify the interactions of two merozoite invasion ligands with their receptors on intact human erythrocytes. BSI assays were performed on unlabelled, free-solution proteins in their native environment, requiring only nanomoles of recombinant protein. This study suggests that BSI can be used to investigate host-parasite protein interactions without the limitations of other assay platforms, and therefore represents a valuable new method to investigate the molecular mechanisms involved in erythrocyte invasion by P. falciparum.

Anti-Plasmodium falciparum invasion ligand antibodies in a low malaria transmission region, Loreto, Peru

BackgroundErythrocyte invasion by Plasmodium falciparum is a complex process that involves two families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins. Antibodies that inhibit merozoite attachment and invasion are believed to be important in mediating naturally acquired immunity and immunity generated by parasite blood stage vaccine candidates. The hypotheses tested in this study were 1) that antibody responses against specific P. falciparum invasion ligands (EBL and PfRh) differ between symptomatic and asymptomatic individuals living in the low-transmission region of the Peruvian Amazon and 2), such antibody responses might have an association, either direct or indirect, with clinical immunity observed in asymptomatically parasitaemic individuals.MethodsELISA was used to assess antibody responses (IgG, IgG1 and IgG3) against recombinant P. falciparum invasion ligands of the EBL (EBA-175, EBA-181, EBA-140) and PfRh families (PfRh1, PfRh2a, PfRh2b, PfRh4 and PfRh5) in 45 individuals infected with P. falciparum from Peruvian Amazon. Individuals were classified as having symptomatic malaria (N=37) or asymptomatic infection (N=8).ResultsAntibody responses against both EBL and PfRh family proteins were significantly higher in asymptomatic compared to symptomatic individuals, demonstrating an association with clinical immunity. Significant differences in the total IgG responses were observed with EBA-175, EBA-181, PfRh2b, and MSP119 (as a control). IgG1 responses against EBA-181, PfRh2a and PfRh2b were significantly higher in the asymptomatic individuals. Total IgG antibody responses against PfRh1, PfRh2a, PfRh2b, PfRh5, EBA-175, EBA-181 and MSP119 proteins were negatively correlated with level of parasitaemia. IgG1 responses against EBA-181, PfRh2a and PfRh2b and IgG3 response for PfRh2a were also negatively correlated with parasitaemia.ConclusionsThese data suggest that falciparum malaria patients who develop clinical immunity (asymptomatic parasitaemia) in a low transmission setting such as the Peruvian Amazon have antibody responses to defined P. falciparum invasion ligand proteins higher than those found in symptomatic (non-immune) patients. While these findings will have to be confirmed by larger studies, these results are consistent with a potential role for one or more of these invasion ligands as a component of an anti-P. falciparum vaccine in low-transmission malaria-endemic regions.

Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.

A new approach to identify human red blood cell receptors for Plasmodium parasites

AbstractMalaria is one of the most significant public health burdens facing the developing world. While there are several Plasmodium species that can cause malaria in humans, the overwhelming majority of malaria mortality is caused by Plasmodium falciparum parasites. The P. falciparum life cycle is complex, but red blood cell invasion is essential for Plasmodium falciparum survival and pathogenesis, and is therefore a topic of particular interest for the development of novel control and intervention strategies. Invasion involves multiple interactions between parasite ligands and their receptors on human red blood cells, and most of these interactions are thought to have overlapping and redundant roles. However, although multiple P. falciparum invasion ligands are known, in very few cases have their red blood cell receptors been identified. This is in part due to the genetic inaccessibility of the erythrocyte, but also in part because cell surface protein‐protein interactions are often of very low affinity, making them hard to identify using standard biochemical approaches. To overcome this, we have used AVEXIS, a systematic protein interaction screening approach designed to detect low affinity extracellular interactions to identify novel red blood cell‐parasite interactions. As a first test of this approach, we produced a library of more than 40 recombinant red blood cell surface protein ectodomains, and screened them against the P. falciparum invasion ligand PfRH5. AVEXIS identified basigin/CD147 as the receptor for PfRH5. Basigin (the Oka blood group antigen) has not previously been implicated as playing a role during P. falciparum invasion. Critically, we showed that basigin was essential for parasite entry in every P. falciparum strain tested to date, as invasion in vitro is potently blocked in all parasite strains tested by soluble basigin receptor ectodomains and by receptor‐specific monoclonal antibodies. The PfRH5‐basigin interaction therefore appears to be an essential and universal entry route for P. falciparum parasites, and is therefore a high priority target for vaccine development. While this work focuses on pathogen‐red blood cell interactions, the AVEXIS approach could equally be applied to identify novel extracellular interactions between other cell types within the human blood system.

High-throughput screens to identify novel interactions between erythrocyte multi-pass receptors and P. falciparum merozoite surface ligands that are involved in invasion

Invasion of erythrocytes by P. falciparum merozoites is a complex multistep process involving a series of molecular recognition events between parasite ligands and their receptors on the erythrocyte surface. Although many potential P. falciparum invasion ligands are known, the erythrocyte receptors they interact with have been identified only for a handful of cases. Studying extracellular binding events between membrane-embedded proteins in vitro is challenging and beyond the reach of standard approaches such as co-immunoprecipitation and yeast two-hybrid screens. These interactions are generally of very low affinity and often require specific motifs generated via post-translational processing; furthermore, amphipathic membrane-spanning proteins are difficult to manipulate biochemically. The Duffy Antigen Receptor for Chemokines (DARC), a multi-pass G-protein coupled erythrocyte receptor is known to be important for invasion by P. vivax but no multi-pass receptor has been established to play a role in P. falciparum invasion. With the aim of identifying such a receptor, we have developed a flow cytometry based, high-throughput strategy that can detect transient, extracellular protein: protein interactions. We are in the process of screening the soluble, recombinant ectodomains of 39 P. falciparum merozoite surface proteins against an expression library of 45 erythrocyte multi-pass surface proteins. We will present our screening strategy and preliminary results.

Differential Antibody Responses to Plasmodium falciparum Invasion Ligand Proteins in Individuals Living in Malaria-Endemic Areas in Brazil and Cameroon

Antibody responses to malaria invasion ligands and proteins on the merozoite surface have been shown to interfere with red cell invasion and correlate with immunity to malaria. The current study is the first to characterize the antibody responses to EBA-140 and EBA-181, Plasmodium falciparum invasion ligands implicated in the alternative pathways of invasion, in age-matched populations of individuals living in endemic areas in both Brazil and Cameroon. Antibody responses to the proteins screened were different between populations. The African individuals reacted strongly with most fragments of these two EBAs, while the majority of the individuals from Mato Grosso, Brazil, reacted weakly and those from the Amazon had elevated responses to these EBA proteins. When compared with the responses against MSP-1(19) and EBA-175, it appeared that the Brazilian population has a variable ability to recognize P. falciparum invasion ligand proteins and that these responses are distinct from the African population.