Multiprotein complex between the GPI-anchored CyRPA with PfRH5 and PfRipr is crucial for Plasmodium falciparum erythrocyte invasion

Erythrocyte invasion by Plasmodium falciparum merozoites is a highly intricate process in which Plasmodium falciparum reticulocyte binding-like homologous protein 5 (PfRH5) is an indispensable parasite ligand that binds with its erythrocyte receptor, Basigin. PfRH5 is a leading blood-stage vaccine candidate because it exhibits limited polymorphisms and elicits potent strain-transcending parasite neutralizing antibodies. However, the mechanism by which it is anchored to the merozoite surface remains unknown because both PfRH5 and the PfRH5-interacting protein (PfRipr) lack transmembrane domains and GPI anchors. Here we have identified a conserved GPI-linked parasite protein, Cysteine-rich protective antigen (CyRPA) as an interacting partner of PfRH5-PfRipr that tethers the PfRH5/PfRipr/CyRPA multiprotein complex on the merozoite surface. CyRPA was demonstrated to be GPI-linked, localized in the micronemes, and essential for erythrocyte invasion. Specific antibodies against the three proteins successfully detected the intact complex in the parasite and coimmunoprecipitated the three interacting partners. Importantly, full-length CyRPA antibodies displayed potent strain-transcending invasion inhibition, as observed for PfRH5. CyRPA does not bind with erythrocytes, suggesting that its parasite neutralizing antibodies likely block its critical interaction with PfRH5-PfRipr, leading to a blockade of erythrocyte invasion. Further, CyRPA and PfRH5 antibody combinations produced synergistic invasion inhibition, suggesting that simultaneous blockade of the PfRH5–Basigin and PfRH5/PfRipr/CyRPA interactions produced an enhanced inhibitory effect. Our discovery of the critical interactions between PfRH5, PfRipr, and the GPI-anchored CyRPA clearly defines the components of the essential PfRH5 adhesion complex for P. falciparum erythrocyte invasion and offers it as a previously unidentified potent target for antimalarial strategies that could abrogate formation of the crucial multiprotein complex.Erythrocyte invasion by Plasmodium falciparum merozoites is crucial for malaria pathogenesis, and thus the parasite has evolved an extensive molecular machinery to ensure invasion through multiple pathways (1–3). The quest to develop successful blood-stage malaria vaccines that efficiently block this process have focused on essential parasite proteins like merozoite surface protein 1 (MSP-1) and apical membrane antigen 1 (AMA-1); however, these are highly polymorphic, unable to elicit strain-transcending neutralizing antibodies, and have thus failed in field trials (4). Among the large repertoire of invasion-related proteins, the family of P. falciparum reticulocyte binding-like homologous (PfRH) proteins have emerged as key determinants of different invasion pathways (2, 3), of which PfRH5 is the only essential conserved parasite ligand (5–8) that elicits potent strain-transcending neutralizing antibodies (9–12). It is localized in the rhoptry and secreted to the merozoite surface during erythrocyte invasion (6). It does not seem to be under immune pressure (9, 13) and is favored to be a leading vaccine candidate. PfRH5 has been shown to interact with another parasite protein, PfRipr (P. falciparum RH5 interacting protein) (14). However, both these proteins lack transmembrane domains as well as a GPI anchor, and thus the mechanism through which PfRH5 is secured on the surface of an invading merozoite to facilitate its functional role during invasion still remains unknown. It is likely that PfRH5 might be attached to the merozoite surface as a complex with other essential proteins other than PfRipr, identification of which could open new therapeutic avenues against malaria.Here we show that PfRH5 and PfRipr interact with a GPI-linked parasite protein, CyRPA (Cysteine-rich protective antigen) (15) to form an essential complex on the surface of an invading merozoite. Individual antibodies against each of the three proteins successfully coimmunoprecipitated all three proteins, confirming their presence as a multiprotein complex. Analysis of the native parasite protein complex by different chromatographic techniques further confirmed that all three protein components coeluted together and were present as a much higher molecular mass species than their individual molecular masses. We also demonstrated that the three proteins are colocalized on the apical surface of the invading merozoite, of which only CyRPA was shown to be GPI-linked. Importantly, antibodies against full-length CyRPA potently blocked erythrocyte invasion by multiple P. falciparum strains, as observed previously only for PfRH5 antibodies (9–12). Because CyRPA does not bind with the erythrocyte surface, it seems that the parasite-neutralizing CyRPA antibodies function by impeding its interaction with PfRH5 or PfRipr. Hence, we have identified and validated a GPI-linked parasite protein, CyRPA, as another essential interacting partner of PfRH5 that is responsible for tethering it to the merozoite surface. Further, we have shown that like PfRH5, CyRPA is a conserved target of potent antibody-mediated blockade of erythrocyte invasion and thus seems to be another highly promising blood-stage vaccine candidate.