Amy K. Bei

Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum

Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor–ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor–ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Oka− erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor–ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

A mitogen-activated protein kinase regulates male gametogenesis and transmission of the malaria parasite Plasmodium berghei

Differentiation of malaria parasites into sexual forms (gametocytes) in the vertebrate host and their subsequent development into gametes in the mosquito vector are crucial steps in the completion of the parasites life cycle and transmission of the disease. The molecular mechanisms that regulate the sexual cycle are poorly understood. Although several signal transduction pathways have been implicated, a clear understanding of the pathways involved has yet to emerge. Here, we show that a Plasmodium berghei homologue of Plasmodium falciparum mitogen‐activated kinase‐2 (Pfmap‐2), a gametocyte‐specific mitogen‐activated protein kinase (MAPK), is required for male gamete formation. Parasites lacking Pbmap‐2 are competent for gametocytogenesis, but exflagellation of male gametocytes, the process that leads to male gamete formation, is almost entirely abolished in mutant parasites. Consistent with this result, transmission of mutant parasites to mosquitoes is grossly impaired. This finding identifies a crucial role for a MAPK pathway in malaria transmission.

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.

Expansion of host cellular niche can drive adaptation of a zoonotic malaria parasite to humans

The macaque malaria parasite Plasmodium knowlesi has recently emerged as an important zoonosis in Southeast Asia. Infections are typically mild but can cause severe disease, achieving parasite densities similar to fatal Plasmodium falciparum infections. Here we show that a primate-adapted P. knowlesi parasite proliferates poorly in human blood due to a strong preference for young red blood cells (RBCs). We establish a continuous in vitro culture system by using human blood enriched for young cells. Mathematical modelling predicts that parasite adaptation for invasion of older RBCs is a likely mechanism leading to high parasite densities in clinical infections. Consistent with this model, we find that P. knowlesi can adapt to invade a wider age range of RBCs, resulting in proliferation in normal human blood. Such cellular niche expansion may increase pathogenesis in humans and will be a key feature to monitor as P. knowlesi emerges in human populations.

A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum

Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry‐based method for simultaneously calculating both the parasitemia and the number of multiply‐infected erythrocytes. Staining with the DNA‐specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion. Am. J. Hematol., 2010.

Functional analysis of epigenetic regulation of tandem RhopH1/clag genes reveals a role in Plasmodium falciparum growth

The Plasmodium RhopH complex is a high molecular weight antigenic complex consisting of three subunits – RhopH1/clag, RhopH2 and RhopH3 – located in the rhoptry secretory organelles of the invasive merozoite. In Plasmodium falciparum RhopH1/clag is encoded by one of five clag genes. Two highly similar paralogous genes, clag 3.1 and clag 3.2, are mutually exclusively expressed. Here we show clonal switching from the clag 3.2 to the clag 3.1 paralogue in vitro. Chromatin immunoprecitation studies suggest that silencing of either clag 3 paralogue is associated with the enrichment of specific histone modifications associated with heterochromatin. We were able to disrupt the clag 3.2 gene, with a drug cassette inserted into the clag 3.2 locus being readily silenced in a position‐dependent and sequence‐independent manner. Activation of this drug cassette by drug selection results in parasites with the clag 3.1 locus silenced and lack full‐length clag 3.1 or 3.2 transcripts. These clag 3‐null parasites demonstrate a significant growth inhibition compared with wild‐type parasites, providing the first genetic evidence for a role for these proteins in efficient parasite proliferation. Epigenetic regulation of these chromosomally proximal members of a multigene family provides a mechanism for both immune evasion and functional diversification.

Molecular analysis of erythrocyte invasion in Plasmodium falciparum isolates from Senegal.

ABSTRACT The human malaria parasite, Plasmodium falciparum, utilizes multiple ligand-receptor interactions for the invasion of human erythrocytes. Members of the reticulocyte binding protein homolog (PfRh) family have been shown to be critical for directing parasites to alternative erythrocyte receptors that define invasion pathways. Recent studies have identified gene amplification, sequence polymorphism, and variant expression of PfRh paralogs as mechanisms underlying discrimination between pathways for invasion. In this study, we find considerable heterogeneity in the invasion profiles of clonal, uncultured P. falciparum parasite isolates from a low-transmission area in Senegal. Molecular analyses revealed minimal variation in protein expression levels of the PfRh ligands, PfRh1, PfRh2a, and PfRh2b, and an absence of gene amplification in these isolates. However, significant sequence polymorphism was found within repeat regions of PfRh1, PfRh2a, and PfRh2b. Furthermore, we identified a large sequence deletion (∼0.58 kb) in the C-terminal region of the PfRh2b gene at a high prevalence in this population. In contrast to findings of earlier studies, we found no associations between specific sequence variants and distinct invasion pathways. Overall these data highlight the importance of region-specific elaborations in PfRh sequence and expression polymorphisms, which has important implications in our understanding of how the malaria parasite responds to polymorphisms in erythrocyte receptors and/or evades the immune system.

Plasmodium falciparum Merozoite Surface Antigen, PfRH5, Elicits Detectable Levels of Invasion-Inhibiting Antibodies in Humans

Plasmodium falciparum is an intracellular protozoan parasite that infects erythrocytes and hepatocytes. The blood stage of its life cycle causes substantial morbidity and mortality associated with millions of infections each year, motivating an intensive search for potential components of a multi-subunit vaccine. In this study, we present data showing that antibodies from natural infections can recognize a recombinant form of the relatively conserved merozoite surface antigen, PfRH5. Furthermore, we performed invasion inhibition assays on clinical isolates and laboratory strains of P. falciparum in the presence of affinity purified antibodies to RH5 and show that these antibodies can inhibit invasion in vitro.

In Vitro Genetic Analysis of an Erythrocyte Determinant of Malaria Infection

Invasion of erythrocytes by Plasmodium falciparum is an obligatory step in the life cycle of the parasite. A major challenge is the unambiguous identification and characterization of host receptors. Because erythrocytes lack nuclei, direct genetic analyses have been limited. In this work, we combined an in vitro erythrocyte culture system, which supports P. falciparum invasion and growth, with lentiviral transduction to knock down gene expression. We genetically demonstrate, in an isogenic background, that glycophorin A is required for efficient strain-specific parasite invasion. We establish the feasibility of in vitro systematic functional analysis of essential erythrocyte determinants of malaria and erythrocyte biology.

Functional diversification between two related Plasmodium falciparum merozoite invasion ligands is determined by changes in the cytoplasmic domain.

The pathogenesis of Plasmodium falciparum depends on efficient invasion into host erythrocytes. Parasite ligands encoded by multi‐gene families interact with erythrocyte receptors. P. falciparum reticulocyte binding protein homologues (PfRhs) are expressed at the apical surface of invasive merozoites and have divergent ectodomains that are postulated to bind different erythrocyte receptors. Variant expression of these paralogues results in the use of alternative invasion pathways. Two PfRh proteins, PfRh2a and PfRh2b, are identical for 2700 N‐terminal amino acids and differ only in a C‐terminal 500 amino acid region, which includes a unique ectodomain, transmembrane domain and cytoplasmic domain. Despite their similarity, PfRh2b is required for a well‐defined invasion pathway while PfRh2a is not required or sufficient for this pathway. Mapping the genomic region encoding these proteins revealed a recombinogenic locus with PfRh2a and PfRh2b in a head‐to‐head orientation. We have generated viable PfRh2a/2b chimeric parasites to identify the regions required for alternative invasion pathway utilization. We find that the differential ability to use these pathways is conferred by the cytoplasmic domains of PfRh2a and PfRh2b, not the ectodomain or transmembrane regions. Our results highlight the importance of the cytoplasmic domain for functional diversification of a major adhesive ligand family in malaria parasites.