A lipid-binding protein mediates rhoptry discharge and invasion in Plasmodium falciparum and Toxoplasma gondii parasites

Abstract

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma, have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium. Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.

Fig. 1

A subset of novel rhoptry surface proteins define a new apical rhoptry sub-compartment. a Comparison of the expression pattern of known rhoptry proteins during the tachyzoite cell cycle in combination with the expression profile of TGGT1_235130 (TgRASP1), TGGT1_315160 (TgRASP2) and TGGT149_306490 (TgRASP3). Data obtained from ToxoDB. b Immunofluorescence assay (IFA) of HA-tagged TGGT1_235130 (TgRASP1) and TGGT1_315160 (TgRASP2) tachyzoites. Insets/higher magnifications: localisation of the proteins overhanging the rhoptry neck (upper panels) and the presence of TgRASP1 in pro-rhoptries (red arrows). Grey arrows indicate mature rhoptries. c Super-resolution microscopy on TgRASP1-HA₃ tachyzoites. Inset: 3D reconstruction of the apical region shows how the rhoptry neck is capped (see X-Y clipping plane). d Schematic representation of the differential permeabilization assay and localisation of ROP5 and ARO. Immunoblots of TgRASP2-HA₃, ROP5, and ARO show that, TgRASP2 is accessible to proteinase K digestion after permeabilization with 1% digitonin (arrow) similarly to the rhoptry surface protein ARO. e Two upper panels: IFAs of TgRASP2-HA₃ and TgRASP3-Ty₃ parasites using anti-HA and anti-Ty antibodies reveal co-localisation of the two proteins. Lower panel: co-localisation of ARO and TgRASP3-Ty₃. TgRASP3 is present at the extremity of the rhoptry, however additional staining in the cytosol is also observed for this protein. Source data are provided as a Source Data file

Fig. 2

TgRASP2 accumulates at the extremity of rhoptries. Immuno-electron microscopy of TgRASP2-HA₃ tachyzoites using an anti-HA antibody. The white arrows point to the gold particles associated with the rhoptries. The white asterisk indicates the rhoptry neck inside the conoid and the red asterisk the accumulation of gold particles at the extremity of a rhoptry. m: micronemes, c: conoid

Fig. 3

Plasmodium falciparum RASP2 expression and localisation in erythrocytic stage. a IFAs on PfRASP2-HA₃ schizonts showing PfRASP2-HA₃ with rhoptry markers. RON4, rhoptry neck, RhopH2, rhoptry bulb. b Comparison of the expression patterns of known rhoptry proteins during the P. falciparum erythrocytic cycle in combination with the expression profile of PF3D7_0210600 (PfRASP2). Real-time transcription data of the P. falciparum 3D7 strain obtained by biosynthetic pyrimidine labeling (PlasmoDB.org). c IFAs of PfRASP2 in free merozoites. d IFAs of invading merozoites. Left, extracellular parasite attached to red blood cell. Middle, at early point of invasion, the RON4 marker detects the moving junction at the posterior end of the intracellular merozoite, while RASP2 remains apical. Right, export of the bulb marker RhopH2 upon invasion. c Pictures of individual merozoites where acquired with a confocal microscope with Airyscan

Fig. 4

Toxoplasma RASP2 depletion impairs parasite invasion. a Strategy for conditional depletion of TgRASP2 using the Tet-OFF system. b Immunoblot of KD-TgRASP2-HA₃ (±ATc). SAG1, loading control. c IFA of KD-TgRASP2-HA₃ shows depletion of RASP2 after 2 days ATc treatment. d Plaque assays of KD-TgRASP2 and complemented cKD-TgRASP2 parasites (±ATc) shows that the strong phenotype induced by TgRASP2 depletion (no plaques) can be rescued by a complementing copy of the gene. CRISPR fitness score was derived from Ref. . e Intracellular replication of TATi_TgRASP2-HA₃ (Ctrl) and KD-TgRASP2-HA₃ ± 48 h ATc. The percentage of vacuoles containing 2, 4, 8, 16 or 32 parasites was determined on 200 vacuoles (n = 3). f Percentage of egressed vacuoles in TATi_TgRASP2-HA₃ (Ctrl) and KD-TgRASP2-HA₃+ATc. Parasite egress was induced 30 h post-invasion by addition of A23187 (3 μM) for 8 min before samples were fixed and processed for IFA (anti-GRA3 antibodies). Egress events (GRA3 in the PV) were quantified on 30 vacuoles. g Gliding assays were performed with KD-TgRASP2 ± ATc 48 h. Trails were revealed by IFA using anti-SAG1 antibodies. h 5 min-invasion assay of TATi_TgRASP2-HA₃ (Ctrl) and KD-TgRASP2-HA₃ parasites (±ATc) shows a strong invasion defect when TgRASP2 is affected. (d, e, f, h) Values represent means ± SD, n = 3, from a representative experiment out of 2 independent assays. Source data are provided as a Source Data file

Fig. 5

P. falciparum RASP2 is necessary for invasion. a Strategy for conditional excision of the pfrasp2 gene using the DiCre system. b PCRs showing efficient excision of the pfrasp2 locus upon addition of rapamycin with primers 3181/2911 (non-excised), 3181/2912 (excised). c IFA on iKO-PfRASP2-HA₃ schizonts ± rapamycin. d Immunoblot on schizont lysates from the p230p DiCre (parental line) and iKO-PfRASP2 mutant line ± rapamycin. PfAldolase, loading control. e Left: Growth curves (parasitaemias) of p230p DiCre (Ctrl) and iKO-PfRASP2 mutant ± rapamycin shows that PfRASP2-depleted parasites are unable to proliferate. Right: Giemsa stainings illustrating the development and reinvasion of iKO-PfRASP2 merozoites (48 h) in the absence of rapamycin and their accumulation at the surface of RBCs in the presence of rapamycin treatment. f Graphical summary of egress data of control (-rapa) and rapamycin-treated iKO-PfRASP2 schizonts following removal of C2. Data collected from 8 control movies (-rapa) and 11 movies in the presence of rapamycin. Number of egress events normalized as a percentage of control (-rapa) considered as 100% egress here. g Invasion rate of Ctrl and iKO-PfRASP2 parasites treated ± rapamycin. Synchronized cultures were treated for 6 h at ring stage with rapamycin or DMSO and were quantified by flow cytometry (100,000 RBCs were analysed for each time point) over 2 cycles (see methods). (e, g) Values represent means ± SD (n = 3) from a representative experiment out of two independent assays. Source data are provided as a Source Data file

Fig. 6

RASP2 plays an essential role in rhoptry secretion. a Immunoblot showing microneme secretion (arrow = processed/secreted TgAMA1) in TATi_TgRASP2-HA₃ (Ctrl) and KD-TgRASP2 parasites. Propranolol induced secretion (Sup induced). TgGRA3, loading control. b Immunoblot of iKO-PfRASP2-HA₃ culture supernatant (secreted proteins) showing microneme secretion (PfAMA1 p44) in control and PfRASP2-depleted parasites. Late schizonts were allowed to egress for 45 min in RPMI. Free merozoites and culture supernatant were separated by centrifugation and processed for Western blot and probed with rabbit anti-PfAMA1. c IFA on E64 or DMSO-treated iKO-PfRASP2-HA₃ parasites ± rapamycin. PfRASP2 depletion does not affect PfAMA1 relocalisation at the surface of merozoites (green arrows). d Left: IFA showing a representative example of an evacuole/rhoptry secretion event in T. gondii (ROP1 staining), Right: Quantification of evacuoles in TATi_TgRASP2-HA₃ (Ctrl) and KD-TgRASP2 parasites (±ATc) (One representative experiment out of three independent assays). e STAT6-P assay on the same lines and the complemented cKD-RASP2 line. f Left: IFA showing two representative examples of a rhoptry secretion event in P. falciparum (RAP2 staining). Right: Quantification of secretion events in iKO-PfRASP2 parasites (±rapamycin). Means ± SD for three independent experiments where >2,500 RBCs were analysed. g Ultrastructure of KD-TgRASP2 + ATc tachyzoites shows no defect in rhoptry positioning. m: micronemes; c: conoid. Source data are provided as a Source Data file

Fig. 7

RASP2 contains lipid binding domains necessary for rhoptry secretion. a Schematic representation of TgRASP2 domains and the TgRASP2con2 recombinant expression construct. b Immunoblots of recombinant TgRASP2con2 and TgRASP2con2^MUT associated or not with liposomes containing phosphatidylethanolamine (PE) and phosphatidylcholine (PC) (proportions 10%: 90%) phosphatidic acid (PA), PE and PC (proportions 30%: 10%: 60%) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), PE and PC (proportions 25%: 10%: 65%). Ctrl, no lipid control; SN, supernatant fraction (unbound); P, pellet fraction (bound protein); Immunoblots were probed with mouse anti-His antibodies. Blots are representative of ≥ three independent experiments. c Quantification of liposome binding assays. p values generated from Student’s t test. Error bars represent ±SD from three or more replicates. d Rhoptry secretion assay with Cpt_WT, ∆PH, L3* and the double mutant ∆PH_L3*. One representative experiment out of three independent assays. Source data are provided as a Source Data file

Fig. 8

Model of RASP2 rhoptry docking/priming function in apicomplexan parasites. Upon response to an unknown signal, RASP2 binds to phospholipids (PA/PIP2) (a). This promotes association of the rhoptry with the parasite plasma membrane to initiate the assembly of the docking/priming machinery of the rhoptry (b), which results in membrane fusion, an essential step before the release of the rhoptry contents into the host cell (c)