Reticulocyte binding protein homologues are key adhesins during erythrocyte invasion by Plasmodium falciparum

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum , invades human erythrocytes through multiple ligand–receptor interactions. The P.  falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1 and PfRh4, members of this protein family, bind to red blood cells and function in merozoite invasion during which they undergo a series of proteolytic cleavage events before and during entry into the host cell. The ectodomain of PfRh1 and PfRh4 are processed to produce fragments consistent with cleavage in the transmembrane domain and released into the supernatant, at about the time of invasion, in a manner consistent with rhomboid protease cleavage. Processing of both PfRh1 and PfRh4, and by extrapolation all membrane-bound members of this protein family, is important for function and release of these proteins on the merozoite surface and they along with EBA-175 are important components of the tight junction, the transient structure that links the erythrocyte via receptor–ligand interactions to the actin–myosin motor in the invading merozoite.     

An EGF-like protein forms a complex with PfRh5 and is required for invasion of human erythrocytes by Plasmodium falciparum

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparumRh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process.     

Reticulocyte-binding protein homologue 1 is required for sialic acid-dependent invasion into human erythrocytes by Plasmodium falciparum

The Apicomplexan parasite responsible for the most virulent form of malaria, Plasmodium falciparum, invades human erythrocytes through multiple ligand-receptor interactions. Some strains of P. falciparum are sensitive to neuraminidase treatment of the host erythrocyte and these parasites have been termed sialic acid-dependent as they utilize receptors containing sialic acid. In contrast, other strains can efficiently invade neuraminidase-treated erythrocytes and hence are sialic acid-independent. The molecular interactions that allow P. falciparum to differentially utilize receptors for merozoite invasion are not understood. The P. falciparum reticulocyte-binding protein homologue (PfRh or PfRBL) family have been implicated in the invasion process but their exact role is unknown. PfRh1, a member of this protein family, appears to be expressed in all parasite lines analysed but there are marked differences in the level of expression between different strains. We have used targeted gene disruption of the PfRh1 gene in P. falciparum to show that the encoded protein is required for sialic acid-dependent invasion of human erythrocytes. The DeltaPfRh1 parasites are able to invade normally; however, they utilize a pattern of ligand-receptor interactions that are more neuraminidase-resistant. Current data suggest a strategy based on the differential function of specific PfRh proteins has evolved to allow P. falciparum parasites to utilize alternative receptors on the erythrocyte surface for evasion of receptor polymorphisms and the host immune system.     

Phenotypic variation of Plasmodium falciparum merozoite proteins directs receptor targeting for invasion of human erythrocytes

The members of the phylum Apicomplexa parasitize a wide range of eukaryotic host cells. Plasmodium falciparum, responsible for the most virulent form of malaria, invades human erythrocytes using several specific and high affinity ligand-receptor interactions that define invasion pathways. We find that members of the P. falciparum reticulocyte-binding homolog protein family, PfRh2a and PfRh2b, are expressed variantly in different lines. Targeted gene disruption shows that PfRh2b mediates a novel invasion pathway and that it functions independently of other related proteins. Phenotypic variation of the PfRh protein family allows P. falciparum to exploit different patterns of receptors on the erythrocyte surface and thereby respond to polymorphisms in erythrocyte receptors and to evade the host immune system.     

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.     

Using Mutagenesis and Structural Biology to Map the Binding Site for the Plasmodium falciparum Merozoite Protein PfRh4 on the Human Immune Adherence Receptor

To survive and replicate within the human host, malaria parasites must invade erythrocytes. Invasion can be mediated by the P. falciparum reticulocyte-binding homologue protein 4 (PfRh4) on the merozoite surface interacting with complement receptor type 1 (CR1, CD35) on the erythrocyte membrane. The PfRh4 attachment site lies within the three N-terminal complement control protein modules (CCPs 1–3) of CR1, which intriguingly also accommodate binding and regulatory sites for the key complement activation-specific proteolytic products, C3b and C4b. One of these regulatory activities is decay-accelerating activity. Although PfRh4 does not impact C3b/C4b binding, it does inhibit this convertase disassociating capability. Here, we have employed ELISA, co-immunoprecipitation, and surface plasmon resonance to demonstrate that CCP 1 contains all the critical residues for PfRh4 interaction. We fine mapped by homologous substitution mutagenesis the PfRh4-binding site on CCP 1 and visualized it with a solution structure of CCPs 1–3 derived by NMR and small angle x-ray scattering. We cross-validated these results by creating an artificial PfRh4-binding site through substitution of putative PfRh4-interacting residues from CCP 1 into their homologous positions within CCP 8; strikingly, this engineered binding site had an ∼30-fold higher affinity for PfRh4 than the native one in CCP 1. These experiments define a candidate site on CR1 by which P. falciparum merozoites gain access to human erythrocytes in a non-sialic acid-dependent pathway of merozoite invasion.     

Evidence that the erythrocyte invasion ligand PfRh2 is a target of protective immunity against Plasmodium falciparum malaria

Abs targeting blood-stage Ags of Plasmodium falciparum are important in acquired immunity to malaria, but major targets remain unclear. The P. falciparum reticulocyte-binding homologs (PfRh) are key ligands used by merozoites during invasion of erythrocytes. PfRh2a and PfRh2b are functionally important members of this family and may be targets of protective immunity, but their potential role in human immunity has not been examined. We expressed eight recombinant proteins covering the entire PfRh2 common region, as well as PfRh2a- and PfRh2b-specific regions. Abs were measured among a cohort of 206 Papua New Guinean children who were followed prospectively for 6 mo for reinfection and malaria. At baseline, Abs were associated with increasing age and active infection. High levels of IgG to all PfRh2 protein constructs were strongly associated with protection from symptomatic malaria and high-density parasitemia. The predominant IgG subclasses were IgG1 and IgG3, with little IgG2 and IgG4 detected. To further understand the significance of PfRh2 as an immune target, we analyzed PfRh2 sequences and found that polymorphisms are concentrated in an N-terminal region of the protein and seem to be under diversifying selection, suggesting immune pressure. Cluster analysis arranged the sequences into two main groups, suggesting that many of the haplotypes identified may be antigenically similar. These findings provide evidence suggesting that PfRh2 is an important target of protective immunity in humans and that Abs act by controlling blood-stage parasitemia and support its potential for vaccine development.     

Cooperativity between Plasmodium falciparum adhesive proteins for invasion into erythrocytes

Plasmodium falciparum is the most virulent of the Plasmodium species infective to humans. Different P. falciparum strains vary in their dependence on erythrocyte receptors for invasion and their ability to switch in their utilization of different receptor repertoires. Members of the reticulocyte-binding protein-like (RBL) family of invasion ligands are postulated to play a central role in defining ligand–receptor interactions, known as invasion pathways. Here we report the targeted gene disruption of PfRh2b and PfRh2a in W2mef, a parasite strain that is heavily dependent on sialic-acid receptors for invasion, and show that the PfRh2b ligand is functional in this parasite background. Like the parental line, parasites lacking either PfRh2a or PfR2b can switch to a sialic acid-independent invasion pathway. However, both of the switched lines exhibit a reduced efficiency for invasion into sialic acid-depleted cells, suggesting a role for both PfRh2b and PfRh2a in invasion via sialic acid-independent receptors. We also find a strong selective pressure for the reconstitution of PfRh2b expression at the expense of PfRh2a. Our results reveal the importance of genetic background in ligand–receptor usage by P. falciparum parasites, and suggest that the co-ordinate expression of PfRh2a, PfRh2b together mediate efficient sialic acid-independent erythrocyte invasion.     

Proteases in malaria-infected red blood cells

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.     

Neutralising antibodies block the function of Rh5/Ripr/CyRPA complex during invasion of Plasmodium falciparum into human erythrocytes

An effective vaccine is a priority for malaria control and elimination. The leading candidate in the Plasmodium falciparum blood stage is PfRh5. PfRh5 assembles into trimeric complex with PfRipr and PfCyRPA in the parasite, and this complex is essential for erythrocyte invasion. In this study, we show that antibodies specific for PfRh5 and PfCyRPA prevent trimeric complex formation. We identify the EGF‐7 domain on PfRipr as a neutralising epitope and demonstrate that antibodies against this region act downstream of complex formation to prevent merozoite invasion. Antibodies against the C‐terminal region of PfRipr were more inhibitory than those against either PfRh5 or PfCyRPA alone, and a combination of antibodies against PfCyRPA and PfRipr acted synergistically to reduce invasion. This study supports prioritisation of PfRipr for development as part of a next‐generation antimalarial vaccine.     

Invasion by P. falciparum merozoites suggests a hierarchy of molecular interactions

Central to the pathology of malaria disease are the repeated cycles of parasite invasion and destruction of human erythrocytes. In Plasmodium falciparum, the most virulent species causing malaria, erythrocyte invasion involves several specific receptor-ligand interactions that direct the pathway used to invade the host cell, with parasites varying in their dependency on these different pathways. Gene disruption of a key invasion ligand in the 3D7 parasite strain, the P. falciparum reticulocyte binding-like homolog 2b (PfRh2b), resulted in the parasite invading via a novel pathway. Here, we show results that suggest the molecular basis for this novel pathway is not due to a molecular switch but is instead mediated by the redeployment of machinery already present in the parent parasite but masked by the dominant role of PfRh2b. This would suggest that interactions directing invasion are organized hierarchically, where silencing of dominant invasion ligands reveal underlying alternative pathways. This provides wild parasites with the ability to adapt to immune-mediated selection or polymorphism in erythrocyte receptors and has implications for the use of invasion-related molecules in candidate vaccines.     

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.     

Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic-basic repeat protein to the surface of Plasmodium falciparum merozoites

Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled-coil. MSP3 also contains a glutamic acid-rich region and a putative leucine zipper sequence at the C-terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid-rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic-basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.     

Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen.     

Analysis of structure and function of the giant protein Pf332 in Plasmodium falciparum

Virulence of Plasmodium falciparum , the most lethal parasitic disease in humans, results in part from adhesiveness and increased rigidity of infected erythrocytes. Pf332 is trafficked to the parasite-infected erythrocyte via Maurer's clefts, structures for protein sorting and export in the host erythrocyte. This protein has a domain similar to the Duffy-binding-like (DBL) domain, which functions by binding to receptors for adherence and invasion. To address structure of the Pf332 DBL domain, we expressed this region, and validated its fold on the basis of the disulphide bond pattern, which conformed to the generic pattern for DBL domains. The modelled structure for Pf332 DBL had differences compared with the erythrocyte-binding region of the αDBL domain of Plasmodium knowlesi Duffy-binding protein (Pkα-DBL). We addressed the function of Pf332 by constructing parasites that either lack expression of the protein or express an altered form. We found no evidence that Pf332 is involved in cytoadhesion or merozoite invasion. Truncation of Pf332 had a significant effect on deformability of the P. falciparum -infected erythrocyte, while loss of the full protein deletion did not. Our data suggest that Pf332 may contribute to the overall deformability of the P. falciparum -infected erythrocyte by anchoring and scaffolding.     

A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1(42)) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Delta1 and MSP9/Delta2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Delta1 and MSP9/Delta2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1(42), the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.     

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.     

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.     

Serine repeat antigen peptides which bind specifically to red blood cells

It has been reported that serine repeat antigen (SERA) binds directly to human erythrocyte membranes, inside-out vesicles and intact mouse erythrocytes. Similarly, mAbs specific against SERA are effective in blocking red blood cell (RBC) invasion by P. falciparum merozoites. Furthermore, the N-terminal recombinant SERA fragment inhibits the merozoite invasion of erythrocyte. In this study of 49 non-overlapping 20-residue-long peptides encompassing the whole SERA protein FCR3 strain, seven peptides having high RBC binding activity were found. Six of these peptides (three from the SERA N-terminal domain) are located in conserved regions and show affinity constants between 150 and 1100 nM, Hill coefficients between 1.5 and 3.0 and 30000-120000 binding sites per cell. Some of these peptides inhibited in vitro merozoite invasion of erythrocyte and intra-erythrocytic development. Residues which are critical in the binding to erythrocytes (in bold face), i.e. 6725 (YLKETNNAISFESNSGSLEKK), 6733 (YALGSDIPEKCDTLASNCFLS), 6737 (YDNILVKMFKTNENNDKSELI), 6746 (DQGNCDTSWIFASKYHLETI), 6754 (YKKVQNLCGDDTADHAVNIVG) and 6762 (NEVSERVHVYHILKHIKDGK), were determined by means of competition assays with high-binding peptide glycine analogues. The identification of peptides which bind to erythrocyte membrane is important in understanding the process of RBC invasion by P. falciparum merozoites.     

Juxtamembrane shedding of Plasmodium falciparum AMA1 is sequence independent and essential, and helps evade invasion-inhibitory antibodies

The malarial life cycle involves repeated rounds of intraerythrocytic replication interspersed by host cell rupture which releases merozoites that rapidly invade fresh erythrocytes. Apical membrane antigen-1 (AMA1) is a merozoite protein that plays a critical role in invasion. Antibodies against AMA1 prevent invasion and can protect against malaria in vivo, so AMA1 is of interest as a malaria vaccine candidate. AMA1 is efficiently shed from the invading parasite surface, predominantly through juxtamembrane cleavage by a membrane-bound protease called SUB2, but also by limited intramembrane cleavage. We have investigated the structural requirements for shedding of Plasmodium falciparum AMA1 (PfAMA1), and the consequences of its inhibition. Mutagenesis of the intramembrane cleavage site by targeted homologous recombination abolished intramembrane cleavage with no effect on parasite viability in vitro. Examination of PfSUB2-mediated shedding of episomally-expressed PfAMA1 revealed that the position of cleavage is determined primarily by its distance from the parasite membrane. Certain mutations at the PfSUB2 cleavage site block shedding, and parasites expressing these non-cleavable forms of PfAMA1 on a background of expression of the wild type gene invade and replicate normally in vitro. The non-cleavable PfAMA1 is also functional in invasion. However - in contrast to the intramembrane cleavage site - mutations that block PfSUB2-mediated shedding could not be stably introduced into the genomic pfama1 locus, indicating that some shedding of PfAMA1 by PfSUB2 is essential. Remarkably, parasites expressing shedding-resistant forms of PfAMA1 exhibit enhanced sensitivity to antibody-mediated inhibition of invasion. Drugs that inhibit PfSUB2 activity should block parasite replication and may also enhance the efficacy of vaccines based on AMA1 and other merozoite surface proteins.