PTRAMP, CSS and Ripr form a conserved complex required for merozoite invasion of Plasmodium species into erythrocytes

Abstract

Invasion of erythrocytes by members of the Plasmodium genus is an essential step of the parasite lifecycle, orchestrated by numerous host-parasite interactions. In P. falciparum Rh5, with PfCyRPA, PfRipr, PfCSS, and PfPTRAMP, forms the essential PCRCR complex which binds basigin on the erythrocyte surface. Rh5 is restricted to P. falciparum and its close relatives; however, PTRAMP, CSS and Ripr orthologs are present across the Plasmodium genus. We investigated PTRAMP, CSS and Ripr orthologs from three species to elucidate common features of the complex. Like P. falciparum, PTRAMP and CSS form a disulfide-linked heterodimer in both P. vivax and P. knowlesi with all three species forming a complex with Ripr by binding its C-terminal region, termed the PTRAMP-CSS-Ripr (PCR) complex. Cross-reactive antibodies targeting the PCR complex differentially inhibit merozoite invasion. The crystal structure of a cross-reactive antibody reveals an inhibitory epitope on the C-terminal tail of PvRipr. Cryo-EM visualization of the P. knowlesi PCR complex confirms predicted models and demonstrates a core invasion scaffold in Plasmodium spp. with implications for vaccines targeting multiple species of malaria-causing parasites.

Fig. 1. PTRAMP, CSS, and Ripr are common to all clades of Plasmodium.

a Comparison of PCRCR orthologs in the Plasmodium genus, showing that PTRAMP, CSS, and Ripr are common to all species, whereas Rh5 is restricted to the Laverania subgenus, and CyRPA is absent from the rodent-infective species (Vinckeia). b AlphaFold 3 predictions of PTRAMP, CSS, and the Ripr tail show a conserved architecture is predicted for each species. For the P. falciparum predicted complex, the previously published structure for PfCSS is superimposed in dark green. Regions that are predicted to be disordered, and therefore have low model confidence, are shown as transparent. The transmembrane domain and signal peptide have been removed from PTRAMP and CSS for clarity. c Domain diagrams for PfPTRAMP, PfCSS and PfRipr showing the predicted N-linked glycosylation sites (blue) that were either mutated (red) or the sequon truncated (gray). Gray regions indicate the stretches of sequence either processed (signal peptides) or removed for recombinant expression, in the case of PfPTRAMP (transmembrane domain and cytoplasmic tail). d SDS-PAGE of glycosylated and non-glycosylated PfPC in both non-reduced (NR) and reduced (R) conditions. Purification was repeated at least three times with similar results. e A representative biolayer interferometry sensorgram of non-glycosylated PfRipr binding to non-glycosylated PfPC showing data (yellow) and 1:1 model best fit (black). Source data for all graphs and uncropped gels are provided as a Source Data file.

Fig. 2. Structure of the intermolecular disulfide bond between PvCSS and PvPTRAMP.

a SDS-PAGE of recombinantly expressed PvPTRAMP, PvCSS and PvPC heterodimer. Purification was repeated at least three times with similar results. b Domain diagram of PvCSS showing the disordered repeat region at the N-terminus. c Crystal structure of PvCSS_115-381(green) and PvPTRAMP_42-53(pink) with the disulfide formed between them in space-filling atomic depiction (yellow). d Detail of the intermolecular disulfide bond between PvCSS and PvPTRAMP. Density is contoured at 1.0 σ and density extends to a range of 1.8 Å. e Structure of PvCSS and PvPTRAMP_42-53, showing the region of the unbiased electron density omit map that was attributed to PvPTRAMP. Density is represented as in d. f The interface between PvPTRAMP_42-53 and PvCSS. All intermolecular hydrogen bonds formed are represented in gray.

Fig. 3. The PvPC heterodimer forms a stable complex with PvRipr.

a–c Representative biolayer interferometry sensorgrams of PvPC, PvPTRAMP, PvCSS, and PvRipr binding assays. Dilution series data are shown in color and 1:1 model best fit is shown in black. d Representative sensorgrams of PvPTRAMP, PvRipr and PvPC binding to PvCyRPA at an analyte concentration of 5 μM. Data could not be reliably fit with a model, and so no best fit has been shown. e Mass distribution of PvPC and PvRipr after pre-incubation as measured by mass photometry. Histogram data are shown in gray and Gaussian curve fit in black. Source data for all graphs are provided as a Source Data file.

Fig. 4. A small region of Ripr is sufficient for PTRAMP-CSS binding in P. falciparum, P. vivax, and P. knowlesi.

a SDS-PAGE of recombinantly expressed PkPTRAMP, PkCSS and PkPC heterodimer. Purification was repeated at least three times with similar results. b–d Representative biolayer interferometry sensorgrams of PkPC, PkPTRAMP, PkCSS and PkRipr binding assays. Dilution series data are shown in color and 1:1 model best fit is shown in black. e Mass distribution of PkPC and PkRipr after pre-incubation as measured by mass photometry. Histogram data are shown in gray and Gaussian curve fit in black. f The ability of Ripr and Ripr truncations to bind to PTRAMP-CSS in P. falciparum, P. vivax and P. knowlesi. The table shows the truncations used and their dissociation constant (K_D, in nM, with standard error of the mean (SEM)) for binding their cognate heterodimer. N.B indicates no binding. Source data for all graphs are provided as a Source Data file.

Fig. 5. Cross-reactive PTRAMP, CSS, and Ripr antibodies exhibit differential inhibition in multiple species of Plasmodium.

a IgG antibodies in human patients with Plasmodium infections. Fold-change peak week one antibody response relative to the seropositivity cut-off (mean of negative controls + 2x standard deviation). b Mouse monoclonal antibodies mapped by binding region. Open text represents antibodies binding both P. vivax and P. knowlesi, and closed text represents cross-reactivity between all three species. Bold lines indicate 4E2 and 2D9 block PvPC binding to PvRipr and that 4H10 blocks PvRipr binding to PvPC. All other antibodies have no effect on PvPC-PvRipr binding and are assumed to be capable of binding the PvPCR complex. c P. knowlesi growth inhibition assay (GIA) of anti-PvPC and anti-PvRipr biologics. The non-inhibitory PfCSS nanobody H2 was included as a negative control. Three independent experiments were performed with mean and SEM shown. Antibodies and nanobodies were tested at a final concentration of 0.5 mg/mL. Data are colored according to antigen (CSS in green, PTRAMP in pink and Ripr in yellow). d GIA dilution series for inhibitory antibodies in P. knowlesi. Growth inhibition (%) is the mean of six independent experiments for 5B3, 2D9, four independent experiments for 5B4 and two independent experiments for 4E2. Error bars represent standard deviation. e GIA dilution series for inhibitory antibodies in P. falciparum. Growth inhibition (%) is the mean of five independent experiments for 5B3, two independent experiments for 4E2, and one independent experiment for 4H10, 2D9 and non-immune IgG. Error bars represent standard deviation. f Ex vivo GIA of P. vivax parasites. Antibodies were tested at a final concentration of 0.5 mg/mL. Anti-Duffy antigen receptor for chemokines (DARC) mouse monoclonal antibody 2C3 was used as a positive control. Data are from six independent experiments. Error bars show mean and SEM. Data are colored as in c. g P. cynomolgi GIA of anti-PvPC and anti-PvRipr antibodies. Antibodies were tested at a final concentration of 0.5 mg/mL. Three independent experiments were performed with mean and SEM shown. Data are colored as in c. Source data are provided as a Source Data file.

Fig. 6. Crystal structure of anti-Ripr mAb 5B3 in complex with PvRipr^EGF7-8.

a Crystal structure of PvRipr^EGF7-8 (yellow) in complex with 5B3 Fab, with heavy chain in dark blue and light chain in light blue. b Sequence alignment of the EGF 7-8 domains from PvRipr, PkRipr, PcRipr and PfRipr. Similarity was predicted using ESPript 3.0 with the “% equivalent” parameter scheme. Identical residues are shown in pink, residues with similar physicochemical properties are in gray and residues with distinct properties in cyan. Mutated N-linked glycosylation sites (Asn767Gln and Asn780Gln) are marked with asterisks. Below is the surface area contribution of each PvRipr EGF 7-8 residue buried by antibody 5B3 as determined by PISA. Residues forming hydrogen bonds of salt bridges with 5B3 are colored black; those involved in van der Waals interactions are gray. c Surface representation of the PvRipr^{EGF 7-8} with the 5B3 epitope colored according to sequence similarity as in b. d, e Detailed views of interactions between 5B3 and polymorphic Ripr residues Glu786 in d and Asn802 in e. Hydrogen bonds and salt bridges are shown as black dashed lines.

Fig. 7. PTRAMP, CSS, and Ripr form a core invasion scaffold in Plasmodium spp.

a Cryo-EM 2D class averages of the PkPCR^tail complex. Addition of the 5B3 Fab fragment shows distinct density at the end of the Ripr tail (white triangle). Inset shows the PkPCR^tail complex colored according to the predicted structure. b AlphaFold 3 predicted structure (left) and cartoon schematic (right) of PkPCR^tail. Transmembrane domains, signal sequence and large disordered regions have been omitted for clarity. Inset shows the alignment of the PvPC (purple and dark green) structure and the PkPC predicted structure (pink and light green). c Erythrocyte binding assays of PkPC, PkRipr and PkPCR. Each color corresponds to one erythrocyte donor. PfRh5 was used as a positive control. Data from three independent experiments with three individual donors are shown with mean and SEM. d Erythrocyte binding assays of PkPC and PvPC using reticulocyte enriched cord blood. PvRBP2b was used as a positive control. Data from three independent experiments with three individual donors are shown with mean and SEM. e Schematic of the PCRCR complex of P. falciparum and PCR complexes of P. vivax, and P. knowlesi. PTRAMP, CSS and Ripr form a conserved three-membered complex that serves as a scaffold for an invasion complex. In P. falciparum this complex involves CyRPA and Rh5. In P. vivax and P. knowlesi this complex likely contains other components (dashed gray) that have yet to be identified, which engage with the erythrocyte membrane via host-cell specific receptors. Source data are provided as a Source Data file.