Sash Lopaticki

Reticulocyte-binding protein homologue 5 - An essential adhesin involved in invasion of human erythrocytes by Plasmodium falciparum

Invasion of erythrocytes is a prerequisite in the life history of the malaria parasite. Members of the reticulocyte-binding homologue family (PfRh) have been implicated in the invasion process and in some cases have been shown to act as adhesins, binding to specific receptors on the erythrocyte surface. We have identified a further, putatively essential, PfRh family member in the most virulent human malaria Plasmodium falciparum, called PfRh5, which binds to an unknown class of glycosylated receptors on the erythrocyte surface. This protein is an atypical PfRh family member, being much smaller than others and lacking a transmembrane and cytosolic region at the C-terminus. This suggests it may be part of a functional protein complex. PfRh5 localises to the rhoptries in merozoites and follows the tight junction during the process of erythrocyte invasion. Furthermore, rabbit immune serum raised against a portion of the ecto-domain, inhibits parasite invasion in vitro. We hypothesise an essential role for the PfRh5 adhesin in erythrocyte selection and commitment to invasion. Given its small size, we believe PfRh5 may prove to be a valuable candidate for inclusion in a multi-component anti-malarial vaccine.

Complement receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand

Plasmodium falciparum is responsible for the most severe form of malaria disease in humans, causing more than 1 million deaths each year. As an obligate intracellular parasite, P. falciparum’s ability to invade erythrocytes is essential for its survival within the human host. P. falciparum invades erythrocytes using multiple host receptor–parasite ligand interactions known as invasion pathways. Here we show that CR1 is the host erythrocyte receptor for PfRh4, a major P. falciparum ligand essential for sialic acid–independent invasion. PfRh4 and CR1 interact directly, with a Kd of 2.9 μM. PfRh4 binding is strongly correlated with the CR1 level on the erythrocyte surface. Parasite invasion via sialic acid–independent pathways is reduced in low-CR1 erythrocytes due to limited availability of this receptor on the surface. Furthermore, soluble CR1 can competitively block binding of PfRh4 to the erythrocyte surface and specifically inhibit sialic acid–independent parasite invasion. These results demonstrate that CR1 is an erythrocyte receptor used by the parasite ligand PfRh4 for P. falciparum invasion.

Reticulocyte and Erythrocyte Binding-Like Proteins Function Cooperatively in Invasion of Human Erythrocytes by Malaria Parasites

ABSTRACT Plasmodium falciparum causes the most severe form of malaria in humans and invades erythrocytes using multiple ligand-receptor interactions. Two important protein families involved in erythrocyte binding are the erythrocyte binding-like (EBL) and the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) proteins. We constructed P. falciparum lines lacking expression of EBL proteins by creating single and double knockouts of the corresponding genes for eba-175, eba-181, and eba-140 and show that the EBL and PfRh proteins function cooperatively, consistent with them playing a similar role in merozoite invasion. We provide evidence that PfRh and EBL proteins functionally interact, as loss of function of EBA-181 ablates the ability of PfRh2a/b protein antibodies to inhibit merozoite invasion. Additionally, loss of function of some ebl genes results in selection for increased transcription of the PfRh family. This provides a rational basis for considering PfRh and EBL proteins for use as a combination vaccine against P. falciparum. We immunized rabbits with combinations of PfRh and EBL proteins to test the ability of antibodies to block merozoite invasion in growth inhibition assays. A combination of EBA-175, PfRh2a/b, and PfRh4 recombinant proteins induced antibodies that potently blocked merozoite invasion. This validates the use of a combination of these ligands as a potential vaccine that would have broad activity against P. falciparum.

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. falciparum Rh5 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.

Role of Plasmepsin V in Export of Diverse Protein Families from the Plasmodium falciparum Exportome

Plasmodium falciparum exports several hundred effector proteins that remodel the host erythrocyte and enable parasites to acquire nutrients, sequester in the circulation and evade immune responses. The majority of exported proteins contain the Plasmodium export element (PEXEL; RxLxE/Q/D) in their N‐terminus, which is proteolytically cleaved in the parasite endoplasmic reticulum by Plasmepsin V, and is necessary for export. Several exported proteins lack a PEXEL or contain noncanonical motifs. Here, we assessed whether Plasmepsin V could process the N‐termini of diverse protein families in P. falciparum. We show that Plasmepsin V cleaves N‐terminal sequences from RIFIN, STEVOR and RESA multigene families, the latter of which contain a relaxed PEXEL (RxLxxE). However, Plasmepsin V does not cleave the N‐terminal sequence of the major exported virulence factor erythrocyte membrane protein 1 (PfEMP1) or the PEXEL‐negative exported proteins SBP‐1 or REX‐2. We probed the substrate specificity of Plasmepsin V and determined that lysine at the PEXEL P3 position, which is present in PfEMP1 and other putatively exported proteins, blocks Plasmepsin V activity. Furthermore, isoleucine at position P1 also blocked Plasmepsin V activity. The specificity of Plasmepsin V is therefore exquisitely confined and we have used this novel information to redefine the predicted P. falciparum PEXEL exportome.

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export, PfEMP1 Display, and Survival of Malaria Parasites

A small molecule inhibitor of the malarial protease Plasmepsin V impairs protein export and cellular remodeling, reducing parasite survival in human erythrocytes.

Plasmodium falciparum uses a key functional site in complement receptor type-1 for invasion of human erythrocytes

The Plasmodium falciparum adhesin PfRh4 binds to complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to complement control protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1s convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between complement regulation and infection.

Erythrocyte-Binding Antigens of Plasmodium falciparum Are Targets of Human Inhibitory Antibodies and Function To Evade Naturally Acquired Immunity

Abs that inhibit Plasmodium falciparum invasion of erythrocytes form an important component of human immunity against malaria, but key target Ags are largely unknown. Phenotypic variation by P. falciparum mediates the evasion of inhibitory Abs, contributing to the capacity of P. falciparum to cause repeat and chronic infections. However, Ags involved in mediating immune evasion have not been defined, and studies of the function of human Abs are limited. In this study, we used novel approaches to determine the importance of P. falciparum erythrocyte-binding Ags (EBAs), which are important invasion ligands, as targets of human invasion-inhibitory Abs and define their role in contributing to immune evasion through variation in function. We evaluated the invasion-inhibitory activity of acquired Abs from malaria-exposed children and adults from Kenya, using P. falciparum with disruption of genes encoding EBA140, EBA175, and EBA181, either individually or combined as EBA140/EBA175 or EBA175/EBA181 double knockouts. Our findings provide important new evidence that variation in the expression and function of the EBAs plays an important role in evasion of acquired Abs and that a substantial amount of phenotypic diversity results from variation in expression of different EBAs that contributes to immune evasion by P. falciparum. All three EBAs were identified as important targets of naturally acquired inhibitory Abs demonstrated by differential inhibition of parental parasites greater than EBA knockout lines. This knowledge will help to advance malaria vaccine development and suggests that multiple invasion ligands need to be targeted to overcome the capacity of P. falciparum for immune evasion.

Plasmodium falciparum merozoite invasion is inhibited by antibodies that target the PfRh2a and b binding domains.

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes.

Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes.

Plasmepsin V, an essential aspartyl protease of malaria parasites, has a key role in the export of effector proteins to parasite-infected erythrocytes. Consequently, it is an important drug target for the two most virulent malaria parasites of humans, Plasmodium falciparum and Plasmodium vivax. We developed a potent inhibitor of plasmepsin V, called WEHI-842, which directly mimics the Plasmodium export element (PEXEL). WEHI-842 inhibits recombinant plasmepsin V with a half-maximal inhibitory concentration of 0.2 nM, efficiently blocks protein export and inhibits parasite growth. We obtained the structure of P. vivax plasmepsin V in complex with WEHI-842 to 2.4-Å resolution, which provides an explanation for the strict requirements for substrate and inhibitor binding. The structure characterizes both a plant-like fold and a malaria-specific helix-turn-helix motif that are likely to be important in cleavage of effector substrates for export.