Parasite, insect cell culture and antibodies

3D7 P. falciparum parasites were obtained from David Walliker, Edinburgh University. Asexual blood stage parasites were grown in in vitro culture as described37.

Sf21 insect cells were cultured in Insect-XPRESS protein-free with l-glutamine (Lonza, 10036636) medium at 28 °C. Expi293F cells were grown in Expi293 expression medium (ThermoFisher) at 37 °C, 8% CO2, 120 r.p.m.

In this study, we used: rat mAb, anti-HA (Roche 3F10, catalogue number 11867423001, lot 47877600); mouse mAbs, 1D9 and 3D8 anti-PfPTRAMP (this study), rat mAb 2D2 anti-PfCSS (this study), mouse mAbs 5B12, 7A6 and 8B9 anti-CyRPA38, 5A9 and 6H2 PfRh510, mouse mAb 1G12 anti-Ripr19, rabbit anti-RON4 polyclonal39; rat pAb KM81 anti-PfCSS (this study); and rabbit pAb R1541 anti-Ripr19.

The mouse mAbs 1D9 and 3D8 that bound PfPTRAMP and the rat mAbs 2D2 mAb and pAb KM81 that bound PfCSS were made at the WEHI Antibody Facility as described in Supplementary Materials and Methods.

The following secondary antibodies labelled with Alexa 488/594 fluorophores (Life Technologies) and HRP antibodies were used: chicken anti-mouse 594 (catalogue number A21201, lot 42099 A), donkey anti-rat 488 (catalogue number A21208, lot 2310102), chicken anti-rabbit 594 (catalogue number A21442, lot 2110863), goat anti-mouse 488 (catalogue number A11001), goat anti-rabbit (catalogue number A11008). Peroxidase affinity pure goat anti-human IgG (H+L) (catalogue number 109-035-088, Jackson Immuno Research).

Transgenic parasites and rhoptry and microneme secretion assay

Transgenic parasite lines were made using CRISPR–Cas9 with methods and oligonucleotides listed in Supplementary Materials and Methods40.

Crosslinking, immunoprecipitation and mass spectrometry analysis

Parasites used for anti-HA antibody immunoprecipitation with and without cross-linking were synchronized and allowed to develop to schizonts; this is described in the Supplementary Materials and Methods.

Live imaging with LLSM

A standard protocol was developed to ensure that parasites were at the same stages for each experiment. Two 30 ml dishes of asynchronous culture were synchronized with 5% sorbitol, as described41. In brief, the culture medium was removed, and the cells were incubated with five volumes of 5% sorbitol in a water bath at 37 °C for 8 min. The sorbitol was then washed off and fresh culture medium added back to the synchronized culture. This synchronization step was repeated 3 days after the first synchronization, and 10 nM rapamycin was added to one of the culture dishes after the second synchronization to induce pfrh5 (3D7–Rh5iKO), pfptramp (3D7–PTRAMPiKO) and pfcss (3D7–CSSiKO) gene deletion in the relevant parasite lines. Two days after the second synchronization, late-stage parasites were isolated from the culture by magnet purification using LS columns attached to MACS MultiStand (Miltenyi Biotec).

Erythrocytes were resuspended at 0.5% haematocrit in RPMI-HEPES supplemented with 0.2% sodium bicarbonate and 5 mM sodium pyruvate (Gibco 11360070). To load uninfected erythrocytes with calcium indicator and stain the plasma membrane, the cells were incubated with 10 μM Fluo-4AM (Invitrogen F14201) for 1 h at 37 °C, and 1.5 μM Di-4-ANEPPDHQ (Invitrogen D36802) membrane marker was added for a further 1 h (refs. 9,16). The stained and loaded erythrocytes were washed three times and resuspended in phenol red free RPMI-HEPES supplemented with 5 mM sodium pyruvate, referred to as pyruvate medium hereafter16.

Purified schizonts were resuspended in culture medium and incubated with 10 nM Mitotracker Red CMXRos (Invitrogen M7512) for 30 min at 37 °C, 5% CO2. The stained schizonts were pelleted, and supernatant was removed before resuspending the schizonts in pyruvate medium. For sample mounting, an acid-washed 5 mm round glass coverslip (Warner Instruments CS-5R) was placed at the bottom of each well in an Ibidi eight-well plate (Ibidi 80826). Each well was then loaded with 200 μl pyruvate medium. Before imaging, 30 μl stained erythrocytes were loaded to a well and left to settle for at least 30 min. After that, 5–10 μl stained schizonts were added to the well and left to settle for around 15 min. A small amount of silicone gel was applied around the coverslip stage of the sample carrier, and a flat head tweezer was used to transfer the coverslip from the well to the sample carrier. The sample carrier was then attached to the microscope such that the coverslip was embedded in the microscope bath filled with 6–8 ml imaging medium that consisted of phenol red free RPMI-HEPES, 10% Albumax, 0.2% sodium bicarbonate, 5 mM sodium pyruvate, 0.25 mM CaCl2 and 10 μM Trolox (Santa Cruz 53188-07-1). Either 5 mg ml−1 D2 anti-CSS nanobody or 1.25 mg ml−1 H8 anti-PTRAMP nanobody was added to the imaging medium for invasion inhibition studies. The imaging experiments were performed on a custom-built LLSM microscope, constructed as outlined in as per licensed plans kindly provided by Janelia Farm Research campus20. Excitation light from either 488 nm or 589 nm diode lasers (MPB Communications) was focused to the back aperture of a 28.6 × 0.7 numerical aperture (NA) excitation objective (Special Optics) via an annular ring of 0.44 inner NA and 0.55 outer NA providing a light sheet with 10 μm length. Fluorescence emission was collected via a 25 × 1.1 NA water dipping objective (Nikon) and detected by either one or two scientific complementary metal–oxide–semiconductor cameras (Hamamatsu Orca Flash 4.0 v2). With the 488 nm excitation, emitted fluorescence was split using a 594 nm dichroic (Semrock) before passing through a LP 594 nm filter (Chroma) on camera A and 525/50 nm (Chroma) filter on camera B. This allowed simultaneous detection of Fluo-4 AM signals by camera B at 500–550 nm range and Di-4-ANEPPDHQ signals by camera A for wavelengths longer than 594 nm. With the 589 nm excitation, emitted fluorescence from Mitotracker Red CMXRos was detected on camera A with the same detection range as previous. All data were acquired in an imaging chamber (Okolabs) set to 36 °C and 5% humidified CO2.

For deconvolution, point spread functions were measured using 100 nm Tetraspeck beads on the surface of a 5 mm coverslip. Data were de-skewed and de-convolved using LLSpy, a Python interface for processing LLSM data. Deconvolution was performed using a Richardson–Lucy algorithm with 15 iterations with the point spread functions generated for each excitation wavelength.

PAM plotting

Parasite–erythrocyte interactions were characterized by plotting the amount of surface contact at each timepoint for each event. The analysis was performed using Imaris (version 9.7.2, Bitplane) with Tracking module. A surface called ‘Erythrocytes’ was first created from the erythrocyte membrane channel with smoothing and absolute intensity setting. The threshold was adjusted either automatically or manually, on some occasions, to obtain an almost continuous surface on the erythrocyte of interest while maintaining the original boundary of the cell. Next, a surface called ‘All parasites’ was created from the parasite channel with smoothing and background subtraction setting. The threshold was adjusted accordingly to achieve reasonable values for parasite surface area (4–9 μm2), and 0.5 μm seed point value was used to split touching parasites. Next, a masked erythrocyte membrane channel was created from the erythrocyte surface by setting the voxel value inside the surface to 1 and outside the surface to 0. From the ‘All parasites’ surface, parasites that interact with the erythrocyte were then selected, by either automated tracking or manual selection, and duplicated into individual surfaces called ‘Parasite 1’, ‘Parasite 2’ and so on. For each parasite, all parts of the surface were selected and then unified and made into a single track. Finally, values of the ‘Intensity Sum’ from the masked erythrocyte membrane channel and the ‘Area’ at each timepoint were extracted from each parasite surface and exported to Microsoft Excel. The ‘Intensity Sum’ values represent the number of voxels in the erythrocyte membrane channel in contact with the parasite surface. The PAM values were then plotted from the Intensity Sum and normalized by the Area.

P. falciparum schizont supernatant and merozoite preparations and analysis

Merozoite and supernatant preparations for SDS–PAGE and immunoblot analysis were performed as previously described40. Synchronized late trophozoite cultures were passed over LD magnetic columns (Miltenyi Biotech) to remove uninfected erythrocytes. Eluted parasites were adjusted to 5 × 106 schizonts per ml and 150 μl added per well of a 96-well flat-bottomed culture dish. The assay dishes were further cultured for 16 h and a representative well smeared for Giemsa staining to ensure either that rupture had occurred normally (control well) or that rupture had been blocked when inhibitors were added. Parasites from each condition were spun at 10,000 × g for 10 min to collect the merozoite pellet and supernatant fractions. Proteins from both fractions were extracted with reducing sample buffer and separated on 4–12% or 3–8% acrylamide gel (NuPAGE, Invitrogen). When inhibitors WM4 and WM382 were at 40 nM and 2.5 nM final concentrations, respectively, a control dish without any protease inhibitor was also included. Parasites were eluted from columns with complete RPMI 1640 culture medium to which the appropriate inhibitor at the same concentration had been added.

Expression and purification of PfCSS, PfPTRAMP, PTRAMP–CSS heterodimer, PfRipr, CyRPA and PfRh5

The gene for the PfPMX cleaved ectodomain of PfPTRAMP (residues 42 to 309) was subcloned into a modified pTRIEX2 vector with N-terminal SUMO and Flag tags followed by a Tobacco etch virus (TEV) protease cleavage site. One potential N-linked glycosylation site at Asn195 was removed by mutation of Thr197 to Ala. The construct was expressed in Sf21 insect cells and secreted into the medium as a soluble protein. The supernatant was purified by ANTI-FLAG M2 Affinity Gel (Merck) and size exclusion chromatography (S200 Increase 10/300 GL, Cytiva). Fractions containing PfPTRAMP were pooled and cleaved with TEV protease for 16 h at 4 °C. His-tagged TEV was removed via NiNTA agarose resin (Qiagen), and PfPTRAMP was further purified via another size exclusion chromatography (S200 Increase 10/300 GL, Cytiva). For biopanning anti-PfPTRAMP nanobodies and their kinetic characterization, a PfPTRAMP (42–309) construct with a C-terminal Avitag was generated and specifically biotinylated42. In addition, a PfPTRAMP construct comprising residues 25 to 309 with a C-terminal His-tag was used for bilayer interferometry binding studies to PfCSS; however, the purification was the same.

The gene for PfCSS (residues 20 to 290) was subcloned into a modified pTRIEX2 vector with a C-terminal Flag tag preceded by a TEV protease cleavage site. The construct was expressed in Sf21 insect cells and purified similarly to PfPTRAMP. The construct used for the alpaca immunization had no potential N-glycosylation sites mutated and was therefore glycosylated. The construct used in binding and crystallization studies had one glycan removed at Asn261, by mutation of Thr263 to Ala.

To generate disulfide-linked PTRAMP–CSS, PfPTRAMP (42–309) and PfCSS (20–290) constructs were co-expressed in Sf21 insect cells and secreted into the medium as a soluble protein. The supernatant was purified by ANTI-FLAG M2 Affinity Gel (Merck) and size exclusion chromatography (S200 Increase 10/300 GL, Cytiva). As both PfPTRAMP and PfCSS constructs contain a Flag tag, some free PfPTRAMP and PfCSS were present along with disulfide-linked PTRAMP–CSS after elution from the ANTI-FLAG M2 Affinity gel; however, they separated well from the disulfide-linked PTRAMP–CSS via size exclusion chromatography due to their differing sizes. Fractions containing PTRAMP–CSS were pooled and cleaved with TEV protease for 16 h at 4 °C. His-tagged TEV was removed via NiNTA agarose resin (Qiagen), and PTRAMP–CSS was further purified via another size exclusion chromatography (S200 Increase 10/300 GL, Cytiva). The purity of PTRAMP–CSS was assessed by SDS–PAGE and shown to be free from monomeric PTRAMP and CSS in non-reducing conditions (Fig. 4b). The PTRAMP–CSS construct used to test D2 nanobody glycan dependency and nanobody–Fc reactivity via western blot had four out of five potential N-linked glycan sites at Asn74, Asn192, Asn234 and Asn261 removed via mutation of the glycan site Thr or Ser to Ala. Mutation of the glycan at Asn283 led to no expression and so was not included. To test binding of nanobodies to PTRAMP–CSS, a biotinylated PTRAMP–CSS protein was generated using the PfPTRAMP (42–309) construct with a C-terminal Avitag.

The gene for PfRipr (residues 20 to 1086) was subcloned into pACGP67a with a C-terminal His tag. The construct was expressed in Sf21 cells and secreted into the medium as soluble protein. The supernatant was dialysed into 20 mM Tris pH 8, 150 mM NaCl. Imidazole was added to 10 mM final concentration, and PfRipr was purified by NiNTA agarose (Qiagen) and eluted in 20 mM Tris pH 8, 150 mM NaCl, 500 mM imidazole. The sample was further purified via size exclusion chromatography, using a S200 Increase 10/300 GL (Cytiva).

The gene for CyRPA (residues 29 to 362) was subcloned into a modified pcDNA3.4-TOPO plasmid with an N-terminal IL-2 signal sequence and a C-terminal Flag tag preceded by a TEV protease cleavage site. Three potential N-linked glycosylation sites at Asn145, Asn322 and Asn338 were removed by mutation of the glycan site Thr or Ser residues to Ala. The construct was expressed via transient transfection of Expi293F cells, and soluble protein was purified from the culture medium in a similar manner to PfPTRAMP described above.

The gene for PMX cleaved PfRh5 (residues 145 to 526) was subcloned into pACGP67a with a C-terminal C-tag. Three potential N-linked glycosylation sites as Asn214, Asn284 and Asn297 were removed by mutation of Thr or Ser residues to Ala. The construct was expressed in Sf21 cells and secreted into the medium as soluble protein. The supernatant was purified by CaptureSelect C-tagXL Affinity Matrix (ThermoFisher) and eluted with 20 mM Tris pH 7.5, 2 M MgCl2. The sample was further purified via size exclusion chromatography, using a S200 Increase 10/300 GL (Cytiva).

Biolayer interferometry studies

Biolayer interferometry experiments were conducted at 25 °C to determine the affinity and epitope bins of selected proteins and nanobodies for PTRAMP–CSS, PfPTRAMP and PfCSS. For protein–protein binding kinetic studies, either PfRipr or PfPTRAMP was diluted into kinetics buffer (PBS, pH 7.4, 0.1% (w/v) BSA, 0.02% (v/v) Tween-20) at 20 μg ml−1 and immobilized onto Anti-Penta-His (His1K) biosensors (Sartorius). Following a 60 s baseline step, biosensors were dipped into wells containing twofold dilution series of either PTRAMP–CSS or PfCSS. Sensors were then dipped back into kinetics buffer to monitor the dissociation rate. For nanobody–PfCSS binding kinetic studies, nanobodies were diluted in kinetics buffer to 5 μg ml−1 and immobilized onto Ni-NTA (NTA) biosensors (Sartorius). Following a 60 s baseline step, biosensors were dipped into wells containing twofold dilution series of either PTRAMP–CSS or PfCSS. Sensors were then dipped back into kinetics buffer to monitor the dissociation rate. For nanobody–PfPTRAMP binding kinetic studies, biotinylated PfPTRAMP or PTRAMP–CSS were immobilized onto High Precision Streptavidin (SAX) biosensors (Sartorius). Following a 60 s baseline step, biosensors were dipped into wells containing twofold dilution series of anti-PfPTRAMP nanobodies.

For competition studies of the anti-PfCSS nanobodies, nanobodies were first diluted in kinetics buffer to 5 μg ml−1 and immobilized onto Ni-NTA (NTA) biosensors (Sartorius). Following a 30 s baseline step, biosensors were dipped into wells containing a negative control nanobody that does not bind the proteins under analysis to quench the sensors. Following another 30 s baseline step, biosensors were dipped into either PfCSS or PTRAMP–CSS. Following a final 30 s baseline step, biosensors were then dipped into a secondary nanobody or PfRipr to assess competition. Due to the moderate affinity of the anti-PfPTRAMP nanobodies, a premix format was employed. Nanobodies or PfRipr were first diluted to 10 μg ml−1 and immobilized onto Anti-Penta-His (His1K) biosensors. Following a 30 s baseline step, biosensors were dipped into wells containing a negative control nanobody that does not bind the proteins under analysis to quench the sensors. Following another 30 s baseline step, biosensors were then dipped into PTRAMP–CSS pre-incubated with a tenfold molar excess of competing secondary nanobody to assess competition.

Kinetics and competition data were analysed using Sartorius’ Data Analysis software 11.0. Kinetic curves were fitted to a 1:1 binding model. Mean kinetic constants reported are the result of two independent experiments. Data presented in Extended Data Fig. 6 represent the per cent of competing nanobody or PfRipr binding compared with the maximum competing nanobody response.

Growth inhibition and flow cytometry of erythrocyte binding and Ca2+ flux

One-cycle growth inhibition and erythrocyte binding assays were performed as described previously11,43. The full methods are described in Supplementary Materials and Methods.

Three-dimensional structure determination of PfCSS–nanobody complexes

For crystallization studies, PTRAMP–CSS and PfCSS alone were mixed with D2 and H2 nanobodies, respectively, in a 1:2 molar ratio, and excess nanobody was purified away via size exclusion chromatography (Superdex 200 Increase 10/300 GL, Cytiva). Complexes were then concentrated to 5 mg ml−1 and mixed 1:1 with mother liquor and set up in hanging or sitting drop crystallization experiments. D2 nanobody–CSS crystallized in 1.6 M ammonium sulfate, 0.1 M sodium chloride and 0.1 M sodium HEPES at pH 7.5 after 1 month and was cryoprotected in 15% (v/v) ethylene glycol. H2–PfCSS crystallized in 0.1 M bis-tris-propane pH 6.0, 17.5% (v/v) PEG3350, 0.2 M sodium malonate in 24 h and was cryoprotected in 15% (v/v) ethylene glycol. Data were collected at the MX2 beamline at the Australian Synchrotron, processed and merged using XDS44 and Aimless45. The positions of the H2 nanobodies in the H2–PfCSS crystal structure were first determined by molecular replacement using the structure of nanobody VHH-α204 from 5HVG with its CDR3 removed46. This solution was then used to build the two PfCSS molecules present in the asymmetric unit via AutoBuild47. This PfCSS structure was then used as a model for molecular replacement in the low-resolution crystal structure of D2 nanobody–CSS, along with VHH-72 from 6WAQ48. PfPTRAMP was not present in the D2–PTRAMP–CSS crystal structure. Presumably, PfPTRAMP and PfCSS dissociated during crystallization, and only D2 nanobody–PfCSS crystallized after 1 month in the high salt crystallization condition. Refinement of the structures was carried out using phenix.refine49 and iterations of refinement using Coot50.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.