2.2. Refolding and purification of His::PfPLSCR

Recovered His::PfPLSCR protein was refolded by rapid dilution into IMAC buffer A [250 mM NaCl, 0.5 mM CaCl₂, 1 mM TCEP, 0.1 % Fos-choline-12 (n-Dodecyl-phosphocholine, Anatrace), 25 mM HEPES pH 7.5], resulting in a final SDS concentration of 0.1 %. Refolded protein was filtered through a 0.45 μm filter prior to immobilised metal affinity chromatography (IMAC) using a HisTRAP™ High Performance column (GE Healthcare), which was equilibrated with IMAC buffer A.

The loaded protein column was washed with several column volumes of wash buffer [IMAC buffer A + 0.4 % SDS] and IMAC buffer A. The protein was eluted in a linear imidazole gradient using IMAC buffer B [IMAC buffer A + 1 M imidazole]. Fractions containing the desired protein were concentrated (Amicon Ultracel 100 K, Millipore) and further purified by size-exclusion chromatography (SEC) using a Superdex S200 10/300 G L column (GE Healthcare) in SEC buffer [250 mM NaCl, 0.5 mM CaCl₂, 1 mM TCEP, 0.1 % FC-12, 25 mM HEPES pH 7.5]. All buffers contained cOmplete™ EDTA-free protease inhibitors (Roche). Purity of the protein was assessed by SDS-PAGE using NuPAGE™ Novex® Bis-Tris gels and blue native PAGE was performed by using the NativePAGE™ Novex® Bis-Tris Gel System and G250 additive (Life Technologies).

2.3. Intrinsic tryptophan fluorescence

Intrinsic tryptophan fluorescence of recombinant protein was measured using a Cary Eclipse Spectrophotometer (Agilent) and Jasco FP-8500 Spectrofluorometer, respectively. Cuvettes were washed with Helmanex™ III and rinsed with ion-free water before each use. Fresh stocks of CaCl₂ and MgCl₂ were prepared using ion-free water for metal ion titration and measurements in reconstitution buffer [100 mM NaCl, 10 mM HEPES, pH 7.5] [30] containing either 2 mM EGTA (for cation free conditions) or 2 mM EGTA and the desired metal ion concentration as calculated by using the Maxchelator programme [https://somapp.ucdmc.ucdavis.edu].

Five μg of purified recombinant protein were used per emission scan (in either duplicate or triplicate measurements per condition and experiment). Intrinsic fluorescence emission spectra were recorded from 310 to 470 nm at 25 °C and an excitation wavelength of 295 nm. Emission and excitation bandwidths were set to 5 nm and 10 nm, respectively. Equilibrium dissociation constants were determined by using the nonlinear regression analysis tool and one-site total binding equation (GraphPad Prism) as described in [28].

2.4. Reconstitution of recombinant His::PfPLSCR into liposomes

The following lipids were obtained from Avanti Polar Lipids to prepare liposomes: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE); 1,2-di-oleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] nickel salt (18:1 DGS-NTA (Ni)); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N-(7-nitro-2-1,3- benzoxadiazol-4-yl) (18:1 NBD-PS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (18:1 NBD-PE).

To prepare liposomes, dried lipid films of either PC only or PC/PE (70:30 % w/w), supplemented with 0.1 % DGS-NTA (Ni) and either 0.25 % NBD-PE (w/w) or 0.25 % NBD-PS (w/w), respectively, were hydrated at 3 mg/mL total lipid in reconstitution buffer and incubated for 1 h at 37 °C under agitation. The emulsion was then sonicated in a sonicator bath for 1 min and subjected to five freeze-thaw cycles. Uniformly sized ULVs (unilamellar vesicles) were obtained using a lipid extruder mounted with a 100 nm membrane (Avanti Polar Lipids).

To prepare proteoliposomes, liposomes were destabilised with n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) at 1:8 M ratio (detergent to lipid) for 15 min at 25 °C prior to the addition of purified His::PfPLSCR (5 μg protein per mg of lipid, adapted from [31]). Aliquots of detergent treated liposomes were taken aside, matching volumes of SEC buffer were added instead of protein and liposome only controls were treated as proteoliposomes which were formed under gentle agitation and incubated at 25 °C for 24 h. Detergent was removed by adding excess amounts (~ 100-fold) of reconstitution buffer and vesicles were collected by ultracentrifugation at 150,000 g (1 h, 25 °C) and resuspended in reconstitution buffer containing 2 mM EGTA and at a lipid concentration of ~ 10 mg/mL.

2.5. Scramblase assay

Based on established methods to characterise PL scramblases [24,[27], [28], [29],31,32], phospholipid translocase activity of recombinant His::PfPLSCR was measured using a Cary Eclipse Spectrophotometer (Agilent) and Jasco FP-8500 Spectrofluorometer, respectively. Stirring cuvettes and bars were rinsed with Helmanex™ III and ion-free water before each use. Five μl of proteoliposome (or liposome only) suspension were diluted in 1 mL of reconstitution buffer containing either 2 mM EGTA or 2 mM EGTA and the desired metal ion concentration and stirred at 25 °C. Emission spectra from 485 to 600 nm were recorded at 25 °C and an excitation wavelength of 470 nm to determine the exact emission maxima of the NBD moieties (533 nm for NBD-PE and 546 nm for NBD-PS). Initial fluorescence was recorded for 100 s prior to the addition of freshly prepared sodium dithionite (in unbuffered 0.5 M Tris) to a final concentration of 30 mM. Fluorescence decay was recorded for the next 400 s and maximal activity was determined by dividing F = fluorescence at Time=500 s by F_max= fluorescence prior to addition of 30 mM sodium dithionite. Traces were fitted using the ‘Plateau followed by one phase decay’ equation in GraphPad Prism.

2.6. Transfection constructs

We used a recently published conditional gene deletion vector [33] to generate pARL PfPLSCR-HA_loxP (Fig. 3A). A targeting fragment composed of the full length Pfplscr coding sequence followed by a triple hemagglutinin tag (HA), as well as two synthetic loxPint modules [34] flanking the recodonised sequence encoding the C-terminal protein region Lys²¹⁷-Lys²⁷⁵ was synthesised (Geneart®) and cloned into pARL FIKK10.1:loxPint: HA (a kind gift from the Treeck lab) by removing the fikk10.1 gene via BglII/PstI.

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Fig. 3

PL scramblase activity of recombinant PfPLSCR in symmetrically labelled vesicles. A) Schematic of the ‘scramblase assay’. B & D) Transbilayer movement of NBD-PS and C & E) NBD-PE in proteoliposomes of different lipid compositions. Scramblase activity (mean ± SD) is presented as the percentage of fluorescence decrease at 500 s. Number of independent experiments is indicated above the error bars. Statistical significance of differences between activities measured in the presence of EGTA and divalent cations was calculated using unpaired t-tests (****, p < 0.0001; ***, p = 0.0001; **, p ≤ 0.005).

Full Pfplscr gene disruption was attempted using CRISPR-Cas9 and by replacing the gene locus with a superfolder green fluorescent protein (sfGFP) expression cassette (Fig. 6A). To generate the donor plasmid pKW003_1022700, primers 1022700 5 F and R were used to amplify the 572 bp 5’ homology flank and primer pair 1022700 3 F and R was used to amplify the 673 bp 3’ homology flank (KOD Hot Start DNA Polymerase, Merck Millipore) which were cloned on either side of the sfGFP expression cassette in pkiwi003 [35] using appropriate restriction sites (Table S1). The pDC2-cam-coCas9-U6.2-hDHFR vector [36] was used to clone in either of the guide RNAs 951969, 952497 and 952530, which were identified by using the web tool CHOPCHOPv2 (http://chopchop.cbu.uib.no [37]). Therefore, two complimentary oligos (of each guide) with 5’ TATT (oligo 1) and 5’ AAAC (oligo 2) overhangs were annealed for ligation into the BbsI digested and dephosphorylated pDC2-cam-coCas9-U6.2-hDHFR vector (a kind gift from the Lee lab).

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Fig. 6

A full gene KO in 3D7 parasites supports the redundancy of PfPLSCR in asexual parasite biology. A) Schematic of the CRISPR-Cas9 mediated KO strategy. Expression of sfGFP is driven under a constitutive BiP promoter. Numbered arrows indicate primers used for genotyping. B) Diagnostic PCR confirms successful replacement of the Pfplscr locus by the sfGFP expression cassette. C) Growth curves of wild-type 3D7 and PfPLSCR KO parasites over two cycles. Parasitemias were averaged from at least three biological replicates using blood from different donors and are presented as mean ± SD. D) Live-cell image of PfPLSCR KO parasites expressing sfGFP throughout asexual parasite development. Parasite nuclei are stained with DAPI (blue).

2.7. Parasite culture and transfection

P. falciparum asexual parasites were cultured in RPMI 1640 medium containing 0.5 % w/v AlbuMax II® (Gibco) and at 4 % haematocrit using human erythrocytes (blood group 0⁺) according to standard procedures [38]. Ring-stage parasites were synchronised using sorbitol treatment. Tightly sychronised DiCre-expressing B11 parasites [39] were transfected with 100 μg of purified pARL PfPLSCR-HA_loxP plasmid and initially selected with 2.5 nM WR99210 (Jacobus Pharmaceuticals) to obtain transgenic parasites harbouring the episomal plasmid. Integration into the genomic locus was achieved by G418 treatment (400 μg/mL) to select for integrants as described in [40].

DiCre-mediated recombination was induced by treating synchronised ring-stage parasites with 100 nM rapamycin (Sigma) in 1 % (v/v) DMSO for 14−16 h to achieve complete excision. In parallel, parasites were mock-treated with 1 % (v/v) DMSO as a negative control. Treated parasites were transferred into 96-well plates at 0.1 % parasitemia (in triplicate) containing fresh red blood cells at 0.1 % haematocrit and were allowed to proceed to the next ring-stage cycle for flow cytometry. Samples for gDNA extraction and PCR amplification, Western blot and immunofluorescence analysis were taken towards the end of the treatment cycle or subsequent cycles.

Ring-stage 3D7 parasites were co-transfected with different combinations of either guide RNA (and Cas9) containing plasmid 951969, 953497 and 953530, as well as with pKW003_1022700 donor plasmid (50 μg each). Drug selection with WR99210 was carried out for 6 days post transfection to ensure sufficient expression of the Cas9 nuclease. Rapid loss of the donor plasmid was assumed in the absence of any selectable marker and sfGFP positive parasites would therefore only be obtained upon successful replacement of the Pfplscr gene with the sfGFP expression cassette.

2.8. P. falciparum gametocyte culture

Asexual parasite cultures were adapted to gametocyte culture media [RPMI 1640 medium containing 5 % pooled human serum, 5 % AlbuMax II®, 4% sodium bicarbonate and 3.5 % Hypoxanthine-Thymidine supplement (Sigma-Aldrich)] prior to the induction of gametocytes. Gametocyte cultures were seeded from synchronised asexual cultures at high ring-stage parasitemia (~ 5–10 %) and 4 % haematocrit and were maintained at 37 °C under 3 % O₂/5 %CO₂/93 % N₂ gas (BOC, UK). Spent culture media was replaced daily for 14 days and cultures were treated with heparin (20 U/mL) on day 4 post induction for four days to kill uncommitted asexual parasites.

2.9. Solubility analysis

Stage-specific infected RBCs were lysed with 0.02 % saponin/PBS. Released parasite material was washed three times with PBS (containing protease inhibitors) prior to hypotonic lysis with 5 mM TrisCl (pH 8.0) for 1 h on ice. The soluble protein fraction was collected by ultracentrifugation at 100,000 g for 30 min at 4 °C. The pellet was resuspended in 100 mM Na₂CO₃ (pH 11.2) to extract peripheral membrane proteins for 1 h on ice. The carbonate-soluble fraction was collected as above and the carbonate-insoluble pellet was needle-passed with 1 % Triton X-100/PBS and incubated at 37 °C for 30 min to extract integral membrane proteins. Triton X-100 soluble and insoluble fractions were separated as above and equal amounts of all fractions were analysed by immunoblotting.

2.10. Western blot analysis

For immunoblots, infected RBCs were lysed and washed as above. Parasite material was resuspended in 1x SDS sample buffer. Extracted proteins were separated on NuPAGE™ Novex® 4–12 % Bis-Tris protein gels in MES buffer (Life Technologies) and transferred onto nitrocellulose membranes using the iBlot® system (Life Technologies). Rabbit anti-HA (clone C29F4, Cell Signaling) was diluted 1:4,000; mouse anti-HA (clone 12CA5, Roche), mouse anti-GFP (clones 7.1 and 13.1, Roche), rabbit anti-ADF1 [41], rabbit anti-F-Actin [42] and mouse anti-Pfs16 (a kind gift from Professor Robert Sauerwein) were diluted 1:1,000 in 5 % skim milk/PBS (w/vol). Horseradish peroxidase-conjugated goat anti-rabbit and goat anti-mouse antibodies were used as secondary antibodies (Jackson IR) and diluted 1:10,000 in 5 % skim milk/PBS.

2.11. Immunofluorescence analysis and imaging

Infected RBCs were fixed with 4 % paraformaldehyde and 0.0075 % glutaraldehyde in PBS for 30 min, permeabilised with 0.1 % Triton X-100/PBS for 10 min, and blocked in 3 % BSA/PBS prior to immuno-labelling with the following primary antibodies diluted in 3 % BSA/PBS: Rabbit anti-HA (1:1,000); mouse anti-HA (1:500), mouse anti-GFP (1:100), rabbit anti-MSP1 (1:500, [43]), rabbit anti-SPP (1:100 [44],), rabbit anti-EXP1 (1:100), mouse anti-Pfs16 (1:1000) and rabbit anti-GAP45 (1:500 [45],). Appropriate Alexa Fluor® conjugated secondary antibodies (Life Technologies) and DAPI (4′,6-diamidino-2-phenylindole) were diluted 1:4,000 in 3 % BSA/PBS. Images were captured using a Nikon Ti Microscope and OrcaFlash4.0 digital camera and were processed with Fiji [46]. Z-stacks were deconvolved using the EpiDEMIC plugin with 80 iterations in Icy [47].

3.2. PfPLSCR is conserved within Plasmodium spp. but not in other Apicomplexa

Orthologs of hPLSCR1 are present in various organisms ranging from fungi and plants to animals (reviewed in [52]). A BLAST analysis against the Eukaryotic Pathogen database (www.eupathdb.org) revealed putative PLSCR proteins across the genus Plasmodium. Protein sequence identities to PfPLSCR range from approx. 68 % for the orthologs of the rodent malaria parasites P. berghei and P. yoelii to 97 % for the putative PLSCR of P. gaboni, a chimpanzee infecting member of the P. falciparum lineage (Fig. 1C). While the N-termini of the Plasmodium spp. PLSCR proteins displayed some variation in length and in the abundance of proline residues, the functional domains of the C-terminal portion appeared to be highly conserved as previously described for other PLSCR orthologs (reviewed in [52]). No putative PLSCR proteins could be identified in the remaining Apicomplexa or unicellular pathogens, including members of the phyla Amoebozoa, Diplomonadida, Kinetoplastida, Oomycetes and Trichomonanida, pointing towards a specific role of the PL scramblase in malaria parasite biology.

The search, however, did identify orthologs in the photosynthetic algae Chromera velia and Vitrella brassicaformis, sharing 24–28 % sequence identity and 37–42 % sequence similarity with the putative PL scramblase of P. falciparum (Fig. S1). The algae represent the closest known relative of apicomplexan parasites and are classified as Chromerida, a marine phylum belonging to an ancient group of unicellular eukaryotes, the Alveolata, which are characterised by flattened, membranous sacs (alveoli) just underneath the plasma membrane [[53], [54], [55]].

3.3. Expression and purification of recombinant PfPLSCR

To biochemically characterise the putative PfPLSCR and validate its potential PL translocase activity, we recombinantly expressed the full-length protein with a N-terminal hexa-histidine affinity tag (His::PfPLSCR) in E. coli BL21(DE3). Several attempts to obtain biologically active His::PfPLSCR using previously described methods for recombinant hPLSCR1 failed [27,30] and required further optimisation to recover functional His::PfPLSCR from inclusion bodies (Fig. 2A). The solubilised protein was refolded by rapid dilution prior to immobilised metal affinity chromatography and gel filtration. Optimal protein stability was achieved by using the zwitterionic surfactant Fos-Choline-12 as determined in a comprehensive detergent screen.

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Fig. 2

PfPLSCR undergoes conformational changes in the presence of metal ions. A) SDS-PAGE of the expression and solubilisation of His::PfPLSCR. The purified recombinant protein runs at the predicted molecular weight of ~ 37 kDa. Pellet and supernatant fractions are labelled as ‘P’ and ‘SN’, respectively. B) BN-PAGE of eluted fractions from the main peak reveals two protein species between the 66 kDa and 146 kDa protein markers (black arrow heads), indicating a possible dimerisation/oligomerisation of His::PfPLSCR. C) Alignment of the putative Ca²⁺ binding regions from different PLSCR1 orthologues. Residues in positions 1, 3, 5, 7, 9 and 12 of the EF-hand-like motif in hPLSCR1 have been proposed to octahedrally coordinate the calcium ion [24] and are shown in bold. Highly conserved residues are highlighted in blue, hydrophobic residues (positions 5 and 7) in green and the strictly conserved phenylalanine residues (position 9) are shown red. D & E) Representative binding curves and calculated affinities (for the shown experiments) of recombinant PfPLSCR for Ca²⁺ and Mg²⁺ ions, respectively. The concentration-dependent decrease of fluorescence at λ 330 nm is plotted as F/F_max with F = fluorescence measured in the presence of cations and F_max= fluorescence under EGTA conditions. A selection of emission spectra is shown in the upper right corner.

A recent study provided first evidence for Ca²⁺-induced oligomerisation of hPLSCR1 which in turn activates PL scrambling [28]. Hence, all steps of inclusion body solubilisation, protein refolding and purification of His::PfPLSCR were carried out in the presence of 0.5 mM Ca²⁺ and Fos-Choline-12. Association of detergent micelles with membrane proteins is known to obscure the elution behavior in size-exclusion chromatography. We therefore performed BN-PAGE to assess the potentially oligomeric state of His::PfPLSCR, which revealed two protein species suggestive of a dimeric and tetrameric form, respectively (Fig. 2B). Further attempts to determine the exact molecular mass of the protein species by size-exclusion chromatography multi-angle light scattering (SEC-MALS) delivered ambiguous results (data not shown), most likely due to the presence of different oligomeric species in the protein solution as implicated by the BN-PAGE of individual fractions.

3.4. PfPLSCR is responsive to metal ions

The hPLSCR1 protein contains a single EF-hand like Ca²⁺ binding loop, which has been shown to bind Ca²⁺ and other divalent cations in a co-ordinated manner, thus inducing conformational changes of the protein [28,29,56]. The unconventional Ca²⁺ binding motif of hPLSCR1 appears to be conserved in the parasite ortholog, including the residues in positions 1 (Asp²⁴⁰), 3 (Asp²⁴²) and 9 (Phe²⁴⁸) (Fig. 2C), all of which have been found to be crucial to the PL translocase activity of hPLSCR1 [24]. A hydrophobic residue in the last position of the Ca²⁺ binding motif in Plasmodium spp. and Chromerida PLSCR orthologs replaces the aspartic acid residue in hPLSCR1 (and other orthologous proteins) (Fig. 2C). Following established methods for the characterisation of the hPLSCR members [25,[27], [28], [29], [30],57], the presence of three tryptophan residues in PfPLSCR enabled the use of intrinsic fluorescence measurements to analyse potential protein conformational changes of PfPLSCR upon binding to Ca²⁺ and Mg²⁺ ions, respectively. Recombinant His::PfPLSCR was therefore titrated with increasing amounts of the respective metal ion until saturation was achieved. Equilibrium dissociation constants were determined by using nonlinear regression analysis as described in [28].

His::PfPLSCR consistently displayed an emission maximum at λ 330 nm. Addition of Ca²⁺ or Mg²⁺ ions resulted in a concentration-dependent decrease of fluorescence intensity with no shift in the emission peak of the protein (Fig. 2D–E). Similar observations were made for hPLSCR1 and hPLSCR2 exhibiting an emission maximum at λ 345 nm [28]. The affinity of the recombinant parasite protein to the two metal ions tested was similar with dissociation constants (K_d) of 6.7 ± 0.6 mM (mean of n = 6 ± SD) for Ca²⁺ and 11.1 ± 1.7 mM (mean of n = 3 ± SD) for Mg²⁺ ions, respectively (Fig. 2D–E). PfPLSCR therefore appears to have an approximately 4-fold higher affinity for Ca²⁺ and a 13-fold higher affinity for Mg²⁺ compared to the values determined for hPLSCR1 [28]. Overall, our observations suggest a functional preservation of the Ca²⁺ binding site in PfPLSCR and imply conformational changes of the protein in response to Ca²⁺ and Mg²⁺, albeit with low affinity as determined by binding constants in the mM range.

3.5. PfPLSCR mediates the transfer of PE and PS in reconstituted proteoliposomes

To investigate the putative PL translocating properties of PfPLSCR, we reconstituted the recombinant protein into liposomes of different lipid compositions and performed ‘scramblase assays’ which allowed us to visualise and monitor PL translocase activity. Addition of sodium dithionate, which cannot cross the artificial membrane, to symmetrically labelled liposomes, reduces fluorescently labelled nitrobenzoxadiazole (NBD) lipids in the outer leaflet, resulting in an approx. 50 % decrease of the initial fluorescence measured. Scrambling activity can be investigated by the ability of the putative PfPLSCR to transfer NBD lipids from the inner to the outer leaflet by which their exposure to the reducing environment will lead to a further decrease in fluorescence (Fig. 3A).

No conclusive PL translocase activity of recombinant His::PfPLSCR could be measured in proteoliposomes composed of PC only and NBD-PS (Figs. 3B & S2A). Some activity, however, was observed if NBD-PE was used as a substrate, leading to a further ~15 % reduction in fluorescence and maximal catalytic function in the presence of 40 mM Ca²⁺ (Figs. 3C & S2B). No PL scrambling could be detected under EGTA conditions, presumably rendering the protein inactive as a consequence of ineffective oligomerisation. Translocase activity could also be observed in the presence of 40 mM Mg²⁺ but with reduced efficiency and comparable to the fluorescence decrease at 10 mM Ca²⁺ (Figs. 3C & S2B). Respective liposomes without PfPLSCR displayed no further decrease in fluorescence beyond the 50 % margin as expected (dotted lines in Fig. S2A-B).

In attempts to mimic the PL composition of the parasite’s membranes, an overall greater catalytic activity was noted in reconstituted proteoliposomes composed of PC and PE (70:30 %), with a further decrease in fluorescence of maximally ~20 % for vesicles labelled with NBD-PS (Figs. 3D & S2C) and ~30 % for NBD-PE containing proteoliposomes (Figs. 3E & S2D). Phospholipid scrambling activity was enhanced by the increase of metal ion concentrations, reaching maximal activity at 20−40 mM Ca²⁺. Mg²⁺ activated PL scrambling could also be observed in these vesicles but once again with lower efficiency. Importantly, no Ca²⁺ induced fluorescence decay (beyond the 50 % margin) was observed for protein-free liposomes composed of PC and PE, excluding the possibility of PE-mediated bilayer destabilisation and liposome ‘leakiness’ (Fig. S2E-F).

These data strongly suggest a metal ion dependent PL translocase function for PfPLSCR with a preference for calcium ions and NBD-PE as a substrate as indicated by maximal activity under these conditions. A recent lipidomic analysis of the total lipid composition in asexual blood stage parasites showed that PC and PE accounted for 50 % of the total lipid content [3]. Inclusion of PE in reconstituted proteoliposomes resulted in an overall increase of His::PfPLSCR mediated PL translocase efficiency, likely due to a more physiological environment allowing for optimal protein function.

3.6. Generation of an inducible PfPLSCR knock out line

Having demonstrated PL translocase activity for PfPLSCR, we undertook a genetic approach to characterise the protein in P. falciparum parasites. We used a previously published genetic system [33] that modifies the endogenous Pfplscr locus by single-crossover homologous recombination using the selection-linked integration (SLI) strategy [40] and generated a conditional knock out (cKO) parasite line expressing C-terminally triple-HA tagged full length PfPLSCR (hereafter referred to as PfPLSCR-HA) under its endogenous promoter. Two synthetic loxPint modules [34] were inserted into the targeting plasmid, allowing for DiCre-mediated excision of the sequence encoding the C-terminal region Lys²¹⁷-Lys²⁷⁵ of the protein in the optimised DiCre recombinase expressing B11 parasite line [39] (Fig. 4A). Transgenic parasites were retrieved after 2 weeks of initial WR99210 selection and a clonal population of integrant parasites was obtained 7 days after neomycin selection using SLI [40]. Successful modification of the Pfplscr locus was confirmed by diagnostic PCR (Fig. 4B) and Western blot analysis giving rise to a ~ 37 kDa protein (doublet) band as predicted for endogenously tagged PfPLSCR-HA (Fig. 4C). The growth rate of PfPLSCR-HA expressing parasites was similar to that of the parental B11 line (Fig. 4D), suggesting no negative side effects of the modification to parasite viability.

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Fig. 4

PfPLSCR-HA is expressed throughout the asexual life cycle and localises to membranous structures in the parasite. A) Schematic of the strategy to generate the conditional KO line PfPLSCR-HA via SLI mediated single-crossover homologous recombination. The targeting plasmid contains the Pfplscr homology region (red), followed by two synthetic loxPint modules flanking the recodonised Pfplscr sequence (red stripes) with a triple-HA tag (blue), T2A skip peptide (gray) and neomycin resistance cassette (yellow). The GFP coding sequence (green) is followed by a stop codon (black bar). Numbered arrows indicate primers used for genotyping. B) Diagnostic PCR confirms successful integration into the Pfplscr locus by amplification of three distinctive fragments at the expected size (1585, 2455 and 1182 bp) and loss of the wild-type band (1396 bp) in the PfPLSCR-HA parasite line. C) Western blot analysis gives rise to a ~ 37 kDa protein band as predicted for PfPLSCR-HA. The endogenously tagged protein is expressed throughout the intra-erythrocytic life cycle as indicated by the different parasite stages (hours post invasion): rings (4 ± 2 h), trophozoites (20 ± 4 h) and schizonts (44 ± 4 h). Anti-ADF (actin depolymerising factor) and anti-F-Actin antibodies are used as loading controls. D) Growth curves of parental B11 and PfPLSCR-HA expressing parasites over two cycles. Parasitemias (as quantified by flow cytometry) were averaged from at least three biological replicates using blood from different donors and are presented as mean ± SD. E) Immunofluorescence analysis of PfPLSCR-HA expressing parasites. The upper panel depicts two ring-stage parasites and co-labelling with anti-SPP (signal peptide peptidase) antibodies as an ER marker. Anti-EXP1 (exported protein 1) delineates the PVM surrounding three trophozoites in a multiply infected red blood cell. Lower two panels show deconvolved co-labelling with antibodies against MSP1 (merozoite surface protein 1) in late blood stage parasites. PfPLSCR-HA co-localises with MSP1 at the plasma membrane in late schizonts. Parasite nuclei are stained with DAPI (blue). Scale bar = 1 μM. F)PfPLSCR-HA remains strongly membrane-associated throughout the intra-erythrocytic life cycle as suggested by sequential extraction of the protein and its presence in the TX-100 insoluble fraction. The blot was cut and probed with anti-ADF as a control for soluble ADF protein and anti-F-Actin as a control for partially membrane-bound parasite actin.

3.7. PfPLSCR-HA localises to different membranous compartments throughout asexual development

The Pfplscr gene is transcribed throughout the intra-erythrocytic life cycle but is maximally expressed in late schizonts stages (40−48 h post invasion, www.PlasmoDB.org). Immunoblot analysis of parasite protein lysates prepared at different timepoints post invasion confirmed the expression of PfPLSCR-HA across the asexual life cycle (Fig. 4C). Immunofluorescence analysis revealed the localisation of the epitope-tagged protein to the periphery of individual merozoites in late schizonts as suggested by co-localisation with the merozoite surface protein MSP1 (Fig. 4E). The protein also localised to a perinuclear region in the early ring stages as evident by partial co-localisation with the ER-resident protein signal peptide peptidase [44], and redistributed to the cytoplasm and vesicular foci in early trophozoite and schizont stages (Fig. 4E). Several attempts to verify the localisation of PfPLSCR-HA at the plasma membrane and other membranous compartments by immuno-EM proved inconclusive, likely due to the inaccessibility of the HA epitope being buried in the membrane. A solubility analysis, however, confirmed a tight membrane association of PfPLSCR-HA throughout the asexual life cycle (Fig. 4F) supporting a possible PL translocase function in the membrane bilayer.

3.8. PfPLSCR is not essential for erythrocyte invasion and asexual development

Investigations into the nature of the C-terminal transmembrane helix of hPLSCR1 confirmed its function in inserting the protein into the bilayer of reconstituted liposomes and revealed an additional role in coordinating the metal ion, thus influencing scramblase activity [25,57]. Two cholesterol-binding sites, composed of Ala²⁹¹, Phe²⁹⁸, Leu²⁹⁹ and Leu²⁹⁹, Phe³⁰², Glu³⁰⁶, respectively have also been identified and proposed to stabilise the C-terminal transmembrane helix in the phospholipid bilayer. The unusual Asp³⁰¹ residue has further been suggested to play a role in scramblase activity [58].

To explore the specific role of PfPLSCR in parasite development, rapamycin induced, DiCre-mediated excision of the ‘floxed’ sequence in the PfPLSCR-HA line was anticipated to generate a mutant scramblase protein lacking the putative NLS, Ca²⁺-binding motif and C-terminal trans-membrane helix (Lys²¹⁷-Lys²⁷⁵ which includes the conserved Asp²⁶⁸ residue) and therefore PL translocase activity, which we hypothesised to be important for host cell invasion and/or intra-erythrocytic development (Fig. 5A). Diagnostic PCRs confirmed successful excision of the locus compared to DMSO-treated PfPLSCR-HA parasites (Fig. 5B). Excision also removed the neo-R gene, enabling expression of the C-terminally truncated scramblase protein in fusion with GFP as evidenced by Western blot analysis (Fig. 5C-D). Live-cell fluorescence of the remaining GFP fusion protein was weak and not clearly distinguishable from background fluorescence but as predicted, plasma membrane localisation in merozoites was no longer observed as visualised by immuno-fluorescence analysis using anti-GFP antibodies in the conditional KO parasites (Fig. 5D). However, no significant growth or invasion defect could be detected for parasites expressing truncated PfPLSCR in the induced KO compared to full length PfPLSCR-HA in mock-treated parasites over a total period of 3 cycles as analysed by flow cytometry (Fig. 5E) or light microscopy of Giemsa-stained blood smears (data not shown). These findings suggest PfPLSCR is dispensable for erythrocyte invasion and asexual parasite development.

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Fig. 5

PL scrambling is not essential for red blood cell invasion and intra-erythrocytic parasite development. A) Schematic of rapamycin induced and DiCre-mediated excision of the floxed sequence in PfPLSCR-HA. B) Diagnostic PCR confirms successful excision of the locus compared to DMSO-treated PfPLSCR-HA parasites. C) The C-terminally truncated PfPLSCR protein is expressed as a GFP fusion protein at the expected molecular weight of ~ 53 kDa in rapamycin-treated parasites. The ~ 27 kDa band is characteristic for GFP only. Anti-ADF antibodies are used as a loading control. D) Plasma membrane association is lost in parasites expressing the truncated PfPLSCR-GFP fusion protein, which distributes throughout the parasite cytosol. E) Growth curves of rapamycin and DMSO-treated parasites over three cycles. Parasitemias were averaged from at least three biological replicates using blood from different donors and are presented as mean ± SD.

To exclude any residual activity of the truncated PfPLSCR protein, we attempted a full KO of the Pfplscr locus using CRISPR-Cas9 by replacing the gene with a super folder GFP (sfGFP) expression cassette (Fig. 6A). Different combinations of the plasmid harbouring the Cas9 expression cassette [36], as well as one of three different guide RNAs tested, were co-transfected with the donor plasmid. One plasmid combination, containing guides 952497 and 952530, both in close proximity to the 5’ targeting flank, resulted in successful parasite recovery after 5 weeks post transfection as evident by sfGFP positive parasites (Fig. 6D). Replacement and loss of the Pfplscr gene with the sfGFP expression cassette was further confirmed by diagnostic PCR (Fig. 6B). A growth analysis of 3D7 wild-type versus PfPLSCR KO parasites revealed no significant differences (Fig. 6C), further corroborating the redundancy of the PL scramblase protein in Plasmodium spp. asexual development.

We thank Matt Child for critically reading the manuscript, Moritz Treeck (Francis Crick Institute, UK) and Marcus Lee (Wellcome Sanger Institute, UK) for providing plasmids, as well as Robert Sauerwein (Radboud UMC), Brendan Crabb (Burnet Institute) and Alan Cowman (Walter & Eliza Hall Institute) for provision of antibodies. We are indebted to Simone Kunzelmann and the Structural Biology STP at the Francis Crick Institute for access to the Jasco FP-8500 Spectrofluorometer and to Marc Morgan at the Imperial College X-ray Crystallography Facility for help with SEC-MALS analysis and crystallisation trials. This research was funded by the Wellcome Trust, Investigator Award to JB (100993/Z/13/Z). For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.