Malaria is caused by Apicomplexan parasites of the genus Plasmodium, of which Plasmodium falciparum is responsible for the majority of severe disease cases in humans. One of the world’s major public health problems, malaria causes an estimated 216 million infections and ~445,000 deaths annually, primarily among young children and pregnant women (WHO, 2018). P. falciparum has a complex life cycle involving stages in the human and mosquito, but disease only occurs during the blood stage, when parasites infect and replicate in human red blood cells (RBCs). As P. falciparum is an obligate intracellular parasite, understanding the molecular determinants of its developmental cycle within RBCs may lead to new therapies. For example, a number of Plasmodium proteins that play key roles during erythrocyte invasion have shown promise as vaccine candidates (Ord et al., 2015; Sack et al., 2017; Salinas et al., 2019). Since natural genetic variation in red cells can influence innate susceptibility to malaria, host erythrocyte factors may also hold potential as therapeutic targets (Taylor and Fairhurst, 2014). The identification and study of such factors, however, has been severely limited by the intractability of mature RBCs, which lack a nucleus and DNA.

P. falciparum invasion of erythrocytes involves a series of coordinated events that unfold rapidly over the course of ~2 min. These events can be divided into three phases: pre-invasion, active invasion, and echinocytosis (Gilson and Crabb, 2009; Weiss et al., 2015). The process is initiated with the rupture of a ‘mother’ parasite, termed a schizont, which releases up to 32 daughter merozoites. During the pre-invasion phase, a free merozoite makes initial contact with the red cell, stimulating shallow deformation of the host cell plasma membrane. Next, the merozoite reorients so its apically-localized organelles are abutting the cell surface. Reorientation is associated with significant membrane deformation and involves interactions between P. falciparum ligands such as the erythrocyte binding antigen (EBA) and reticulocyte binding-like homologues (Rh) family proteins, and receptors on the red cell surface (Gilson and Crabb, 2009; Paul et al., 2015; Riglar et al., 2011; Tham et al., 2012; Weiss et al., 2015). Several ligand and receptor pairs have been shown to act at this stage, often in a strain-specific manner (e.g. PfEBA-175 and GYPA; PfEBA-181 and GYPB PfEBA-140 and GYPC; and PfRh4 and CR1), but experimental data suggest their roles in apical reorientation and host cell deformation are largely functionally redundant (Tham et al., 2012).

The only receptor-ligand interaction known to be essential during the pre-invasion phase involves basigin and PfRH5, which exists in a complex with PfRipr and CyRPA (Chen et al., 2011; Crosnier et al., 2011; Dreyer et al., 2012; Reddy et al., 2015). Binding of the PfRH5 complex to basigin is required for discharge of the rhoptry organelles into the invaded cell and is associated with a calcium spike, potentially due to formation of a pore at the erythrocyte surface (Volz et al., 2016; Weiss et al., 2015). Blocking the interaction between PfRH5 and basigin with specific antibodies prevents invasion (Crosnier et al., 2011; Patel et al., 2013).

Discharge of the rhoptry organelles heralds the start of active invasion. Among the proteins injected from the rhoptries are those of the RON complex (RON 2, RON 4 and RON 5), which together form a receptor for binding by the PfAMA1 protein localized on the merozoite surface (Alexander et al., 2006; Alexander et al., 2005; Richard et al., 2010). The interaction between PfAMA1 and the RON complex forms a moving junction between the parasite and host cell that is believed to be entirely parasite-derived (Besteiro et al., 2011; Besteiro et al., 2009; Harvey et al., 2014; Koch and Baum, 2016). The moving junction provides an anchoring point for the merozoite to actively invade using its own actinomyosin motor; inhibiting the interaction between PfAMA1 and RON prevents invasion (Richard et al., 2010; Srinivasan et al., 2011; Yap et al., 2014).

As invasion proceeds, a parasitophorous vacuole is formed from components of the host cell membrane and rhoptries, yielding a protective niche for development of the new daughter parasite. The third phase, echinocytosis, is a transient period of cell dehydration and shrinkage observed after invasion; current evidence suggests it is triggered by discharge of the rhoptry contents, prior to and independent of active invasion. Echinocytosis is inhibited by reagents that prevent rhoptry discharge, such as antibodies targeting basigin or PfRH5 (Weiss et al., 2015).

Most host factors known to play a role in P. falciparum invasion have been identified based on their ability to bind to established invasion ligands or from studies of rare natural mutants (Bei and Duraisingh, 2012). Given the inherent intractability of mature RBCs, which lack a nucleus and DNA, the use of genetic approaches to identify and characterize malaria host factors presents a logistical challenge (Egan, 2018). Recently, an shRNA-based forward genetic screen using cultured red cells (cRBCs) derived ex-vivo from primary human hematopoietic stem/progenitor cells (HSPCs) identified erythrocyte CD55 (aka DAF) as a critical host factor for P. falciparum invasion (Egan et al., 2015). A 70 kD extracellular glycoprotein anchored to the red cell membrane by a glycosylphosphatidylinositol (GPI) linkage, CD55 is broadly distributed in different tissues and secretions, including blood cells (Cooling, 2015; Storry et al., 2010). On erythrocytes, CD55 acts as a complement regulatory protein to prevent complement-mediated damage. On epithelial cells, it has been shown to act as a receptor for Group B coxsackie virus and Dr+ E. coli (Cooling, 2015; Coyne and Bergelson, 2006).

We have shown that P. falciparum invasion efficiency was reduced by ~50% in CD55-knockdown cRBCs, and natural CD55-null erythrocytes from two rare donors with the Inab phenotype were resistant to invasion (Egan et al., 2015). Importantly, the requirement for CD55 was strain-transcendent, suggesting that it plays a conserved role in P. falciparum invasion. However, the precise function of CD55 during invasion and how it may interface with established ligands or receptors is unknown.

In this study, we investigated the functional role of CD55 during P. falciparum invasion using CRISPR-Cas9 genome editing, antibody-based inhibition and live cell microscopy. We found that CD55 plays a critical role during P. falciparum invasion of mature erythrocytes, where it is specifically required for parasite internalization. As a host factor that acts after discharge of the parasite’s rhoptry contents, CD55 plays a unique role relative to other receptors required for invasion, providing a crucial link between the ligand-receptor interactions important for adhesion and deformation and effective internalization.

A comprehensive understanding of the molecular interactions required for P. falciparum invasion has been limited by the absence of a robust genetic system to study red cells, which are terminally differentiated and lack a nucleus and DNA. Previously, an RNAi-based forward genetic screen in cultured red cells derived from HSPCs identified two new candidate host factors for invasion: CD44 and CD55 (Egan et al., 2015). In this study, we used a combination of genetics and inhibitory antibodies to determine the precise steps of P. falciparum invasion during which CD55 functions. We have demonstrated that CD55 is specifically required for merozoite internalization, and plays a unique role relative to the other host receptors known to act during invasion.

Prior work has shown that the efficiency of P. falciparum infection is reduced by ~50% in CD55-deficient cRBCs where expression is downregulated using shRNAs. While experiments using erythrocytes from two rare, CD55-null donors suggested that CD55 is essential for P. falciparum invasion, this has never been demonstrated genetically. A major roadblock to such experiments has been the technical challenges associated with CRISPR-Cas9 genome editing in primary human hematopoietic stem cells, including cytotoxicity from nucleic acids and low rates of transfection (Hendel et al., 2015). In this study, we showed for the first time that fully mature, CD55-null cRBCs can be generated efficiently from primary human HSPCs using CRISPR-Cas9 genome editing. Using isogenic wild-type and mutant cells, we have demonstrated that CD55-null cRBCs are resistant to P. falciparum invasion, confirming that CD55 is an essential host factor for P. falciparum.

Here, we demonstrated the feasibility and benefits of generating truly mature erythrocytes ex-vivo for the study of malaria host factors, as these cells closely mimic the target cell for P. falciparum in the human bloodstream. Our approach involving the co-delivery of two chemically modified sgRNAs together with Cas9 as a ribonucleoprotein complex has been shown to be an effective strategy for CRISPR-based gene knockout in primary HSPCs and T cells (Hendel et al., 2015). This method obviates the toxicity associated with plasmid delivery, minimizes off-target activity, and improves sgRNA stability. Combined with our ex vivo erythropoiesis culture system, this method can efficiently generate terminally differentiated, enucleated, CD71-low red cells with a high rate of gene knock out that can be used to study host genetic determinants for P. falciparum in an isogenic background.

We observed that the reliance of P. falciparum on CD55 for invasion increased significantly as enucleated cRBCs matured into erythrocytes, likely reflecting the substantial changes in protein abundance that occur during terminal maturation of human red cells, including for proteins known to act as receptors, such as basigin and CR1 (Gautier et al., 2016; Hu et al., 2010). The efficiency of P. falciparum invasion is further influenced by the deformability of the red cell membrane (Tiffert et al., 2005), a biophysical property that also changes as red cell progenitors proceed through enucleation and final maturation (Giarratana et al., 2005). As clinical malaria is primarily a disease of mature, peripheral blood erythrocytes, our results showing that terminal red cell maturation modifies the requirement for CD55 during invasion highlights the potential drawbacks of using cell lines to study host factors for P. falciparum. While considered erythroid in nature, immortalized cell lines such as JK-1, EJ and BEL-A have low rates of enucleation and terminal maturation (Kanjee et al., 2017; Satchwell et al., 2019; Scully et al., 2019), suggesting their utility for genetic experiments on P. falciparum invasion may be limited by incomplete phenotypes.

Antibodies targeting a variety of specific host receptors for P. falciparum have been shown to have invasion inhibitory activity, including those against GYPA, CR1, and basigin (Crosnier et al., 2011; Pasvol et al., 1989; Spadafora et al., 2010). While we found that individual monoclonal antibodies against three distinct SCR domains of CD55 had no discernable effect on P. falciparum growth, in combination they had potent dose-dependent inhibitory activity, suggesting CD55’s role in invasion is not restricted to a specific SCR domain. Our demonstration that both the polyclonal anti-CD55 antibody and anti-CD55 Fab fragments can inhibit P. falciparum growth aligns with the results from our genetic studies, and corroborates the conclusion that CD55 is a critical host factor for P. falciparum. In contrast to CR1, where antibodies are only inhibitory in the absence of red cell sialic acid (Spadafora et al., 2010), anti-CD55 antibody blocked both sialic-acid-dependent and -independent invasion. This finding is consistent with prior studies showing that the requirement for CD55 in P. falciparum invasion is strain-transcendent (Egan et al., 2015), and implies a model where CD55 acts distinctly from CR1 and the other alternative, strain-specific receptors.

Given the inherent limitations associated with studying invasion on a population scale in longer term assays, live microscopy has been increasingly employed to visualize individual P. falciparum invasion events in real time. Analysis of the morphology and kinetics of discrete invasion steps in the context of blocking antibodies or soluble proteins has contributed to a model describing the molecular events that occur during invasion (Volz et al., 2016; Weiss et al., 2015). Our live microscopy experiments have added a new dimension to this model by revealing that merozoite internalization is inhibited in the presence of anti-CD55 antibody or with CD55-null erythrocytes, demonstrating for the first time that CD55 is specifically required for parasite entry. Where and how does CD55 act in relation to the P. falciparum ligands and erythrocyte receptors known to function during invasion? We observed no impact of the anti-CD55 antibody on the number of merozoite contacts with the RBC, pre-invasion kinetics, or merozoite-induced erythrocyte deformability, findings that were validated using CD55-null erythrocytes from a rare donor. These results suggest that CD55 functions distinctly from the ‘alternative’ receptors required for apical reorientation and red cell deformation (e.g. glycophorins and CR1), as blocking interactions between these receptors and their ligands strongly inhibits merozoite-induced deformability (Weiss et al., 2015).

Echinocytosis is a phenomenon of transient red cell shrinkage that commences soon after merozoite internalization. Current evidence suggests that it is stimulated by changes in the red cell cytoplasm that occur once an irreversibly attached merozoite discharges its rhoptry contents into the erythrocyte (Volz et al., 2016; Weiss et al., 2015). Echinocytosis is inhibited by antibodies that block the interaction between PfRH5 and basigin, implying that this interaction is necessary for rhoptry discharge (Weiss et al., 2015). These blocking antibodies also inhibit Ca²⁺ flux into the erythrocyte, possibly reflecting formation of a pore at the cellular interface when PfRH5 and basigin interact (Volz et al., 2016). The glycophorins and CR1 act early in the pre-invasion phase during apical reorientation, and blocking their interactions with ligands using genetics, enzyme treatments, or antibody blockade also inhibits echinocytosis (Volz et al., 2016; Weiss et al., 2015). In contrast, echinocytosis is not prevented by treatment with the actin polymerization inhibitor cyt-D or reagents that block the interaction of PfAMA1 and the RON complex. Together, these findings demonstrate that the stimulus for echinocytosis occurs after the interaction of PfRH5 with basigin, but before establishment of the moving junction.

Our observation that anti-CD55 antibody had no effect on the efficiency of merozoite-induced echinocytosis suggests that CD55 acts after discharge of the parasite’s rhoptry organelles. Consistent with this idea, we found that merozoites could stimulate CD55-null RBCs to undergo echinocytosis, which should not happen if rhoptry discharge were prevented. Since the overall rate of echinocytosis was highly variable in the previously cryopreserved WT and CD55-null RBCs used in this study and the anti-CD55 antibody only partially inhibits invasion, we also sought to obtain direct evidence for rhoptry secretion in the absence of CD55. Using immunofluorescence assays, we showed that the rhoptry protein RAP1 localized in a pattern consistent with secreted protein in merozoites attached to both WT and CD55-null RBCs, indicating that rhoptry discharge had occurred. Together, these findings demonstrate that CD55 acts after discharge of the rhoptries, highlighting its role as distinct from basigin and the other established host receptors for P. falciparum invasion.

Merozoite internalization requires the formation of a moving junction between the cell membranes of the invading merozoite and the erythrocyte that moves posteriorly down the parasite as it invades. The current model of the moving junction involves interactions between PfAMA1 expressed on the merozoite surface and the PfRON complex inserted into the red cell membrane, independent of any host-encoded receptors (Alexander et al., 2006; Alexander et al., 2005; Besteiro et al., 2009; Richard et al., 2010; Riglar et al., 2011; Srinivasan et al., 2011). In addition to its key role in internalization, the moving junction has also been proposed to be important for formation of the parasitophorous vacuole (PV) (Yap et al., 2014).

Could CD55 function as a component of the moving junction during invasion? We observed that PfAMA1 and PfRON4 were almost always co-localized at the cellular interface for merozoites attempting to invade WT cells, whereas this was significantly less common for merozoites attempting to invade CD55-null erythrocytes. For the minority of merozoites that attached to CD55-null erythrocytes and had PfAMA1 co-localized with RON at the cellular interface, none were observed to have progressed past early invasion, unlike those attached to WT cells. Together, these findings support a model in which CD55 is required for progression of the moving junction, either directly or indirectly. Consistent with this model, irreversible attachment of merozoites to RBCs was completely inhibited in the presence of the junction-blocking R1 peptide, but only moderately inhibited in the absence of CD55, suggesting that CD55 is required not for formation of the moving junction, but for its stability or progression. Additionally, in our immunofluorescence assays of RAP1 localization, we observed a few instances where RAP1 was spread across the RBC surface only in CD55-null RBCs, a phenotype that has been described previously for merozoites attached to RBCs in the presence of R1, and has been speculated to reflect impaired sealing of the moving junction (Riglar et al., 2011). Future studies involving higher resolution microscopy such as cryo-EM will be necessary to better characterize how absence of CD55 may impact the moving junction or architecture of the PV after invasion.

Our findings showing that the function of CD55 during invasion can be narrowed to the internalization step are consistent with a model where CD55 acts distinctly from the other known receptors for invasion, potentially by interacting with a specific parasite ligand. As there is a precedent for CD55 on epithelial cells to act as a pathogen receptor that transmits signals to the host cell (Coyne and Bergelson, 2006), it is tantalizing to hypothesize that this molecule functions similarly in P. falciparum invasion of erythrocytes. Ultimately, biochemical identification of parasite and erythrocyte interaction partners of CD55 will yield important additional insights into the molecular function of CD55 during merozoite internalization. Given the complexity of P. falciparum invasion and the unique and essential role of CD55 relative to other established receptors, targeting its activity or interaction partners in novel intervention strategies may enhance the effectiveness of future therapies or vaccines for malaria.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Bikash Shakya

   Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9100-0660

2. Saurabh D Patel

   Zuckerman Institute, Columbia University, New York City, United States

   Contribution

   Conceptualization, Resources, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5738-3175

3. Yoshihiko Tani

   Japanese Red Cross Osaka Blood Center, Osaka, Japan

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

4. Elizabeth S Egan

   Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   eegan@stanford.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2112-7700

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