Preparation of PfMSA180 recombinant proteins
Construct 1: The DNA for the N-terminal recombinant protein construct 1 (C1) was cloned in the pET24b vector using NdeI and XhoI sites. The selected plasmid was inserted into E. coli ArcticExpress cells and expression of a soluble protein was induced following treatment with 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG). Following induction, bacteria were harvested by centrifugation and the cell pellet was lysed by sonication. The protein was purified from the supernatant fraction of the lysate by immobilized metal affinity chromatography (IMAC) using a Ni–NTA column and eluted with increasing imidazole concentration. The protein was purified by size exclusion chromatography using a S200 16/600 column (GE Healthcare).
Constructs 2, 3 and 4: The DNA for construct 2 (C2) and 3 (C3) was cloned in the pET24b vector using NdeI and XhoI sites, while the C-terminal construct 4 (C4) was cloned in pET24b vector using NheI and XhoI sites. E. coli Shuffle 26 cells were transformed with the C2 and C3 plasmids while the E. coli BLR cells were transformed with the C4 plasmid. Following maximal expression induced by treatment with 1 mM IPTG, the recombinant proteins (C2, C3, C4) were expressed in the form of inclusion bodies (IB). The bacterial pellet was lysed by sonication and the inclusion bodies were prepared and washed thoroughly with buffers containing Triton X-100 and Urea. Washed IBs were solubilised in a buffer containing 6 M guanidine hydrochloride and the proteins were purified by IMAC using Ni–NTA columns and elution done with increasing imidazole concentration. The IMAC purified proteins were refolded in a Tris-based buffer (55 mM Tris pH 8.2, 264 mM NaCl, 11 mM KCl, 2.2 mM MgCl2, 2.2 mM CaCl2, 550 mM L-Arginine, 440 mM sucrose) and dialysed against a Tris-based buffer (55 mM Tris pH 8.2, 10.56 mM NaCl, 0.44 mM KCl, 5% Glycerol). All three proteins were purified to homogeneity by size exclusion chromatography using a S200 16/600 column (GE Healthcare).
Animal immunization and antibody purification
BALB/c mice were injected with recombinant antigen to raise specific antibodies. For priming (day 0), 25 µg of antigen formulated with complete Freund’s adjuvant (CFA) (Sigma, St. Louis, MO) was injected intramuscularly, then two booster immunizations with 25 µg of antigen formulated with incomplete Freund’s adjuvant (IFA) (Sigma, St. Louis, MO) were injected on days 28 and 56. Serum was collected on day zero before priming (pre-bleed), and on days 14, 42 and 70.
New Zealand White (NZW) rabbits were immunized intramuscularly with all four recombinant antigens. For priming (day 0), 100 µg of antigen formulated with CFA was injected, then booster immunizations with 100 µg of antigen formulated with IFA were given on days 28 and 56. Serum was collected on day zero before priming (pre-bleed), and at days 14, 42 and 7034.
Serum immunoglobulin G (IgG) was purified from the rabbit sera collected on Day 70 using a protein G-sepharose column (GE Healthcare). Serum was mixed with binding buffer (20 mM phosphate buffer, pH 7.0) in a 1:1 ratio and loaded on to a pre-equilibrated IgG column (1 ml packed column volume). After the diluted serum was loaded, the column was washed with binding buffer (30 times the column volume). IgG was eluted with elution buffer (0.1 M glycine–HCl, pH 2.7) and neutralised by addition of 5% neutralisation buffer (1 M Tris–HCl, pH 9.0). The purified IgG was pooled, concentrated (10 kDa Centricon, Merck) and dialysed against incomplete RPMI 1640 medium. The purified IgG was filtered with 0.2 µm syringe filters and stored at -80 °C until used for invasion inhibition assays34.
Erythrocyte binding assay
10 µg of recombinant proteins were incubated with 100 µl of packed erythrocytes in incomplete RPMI 1640 (containing 360 μM hypoxanthine, 24 mM sodium bicarbonate and 10 μg/ml gentamycin) at 37 °C for 1 h. The erythrocytes were washed and incubated with primary antibody against the recombinant antigens at 37 °C for 1 h. The cells were washed and incubated with Alexafluor-594 (Sigma) conjugated secondary antibodies. The cells were washed again and protein binding was measured by flow cytometry (BD FACS Aria)35.
Parasite culture
The P. falciparum 3D7 and II-3 DiCre-expressing parasite lines were cultured in vitro in complete RPMI 1640 medium (Invitrogen) containing 0.5% Albumax, 360 μM hypoxanthine, 24 mM sodium bicarbonate and 10 μg/ml gentamycin, in human O positive erythrocytes at 2% hematocrit in mixed gas (5% CO2, 5% O2, 90% N2) at 37 °C as described previously36. The parasites were synchronised by sorbitol37 and percoll treatments38.
Growth inhibition assay (GIA)
The assay was performed as per the protocol described earlier34,39. Synchronized late-stage schizont parasites at 2% hematocrit and 0.3% initial parasitemia were incubated with purified IgG at 10.0 mg/ml (CyRPA was tested at 2.5 mg/ml). After one-cycle (40–44 h) of invasion, parasites were stained with ethidium-bromide and parasitemia was measured by flow cytometry (BD FACS Aria). Inhibition of parasite growth in the presence of IgG was calculated relative to the growth of parasites incubated with the IgG purified from pre-immune sera.
Immunoblot analysis of parasite lysate
P. falciparum synchronised late-stage schizonts were harvested by centrifugation. Erythrocytes were lysed with 0.05% (w/v) saponin before the parasite pellet was lysed in RIPA buffer (Sigma) with 2X protease inhibitor cocktail (Roche) on ice for 2–3 h. The parasite lysate was clarified by centrifugation (16,000 g, 30 min) and proteins in the supernatant were resolved by 8% SDS-PAGE and transferred on to 0.45 µm nitrocellulose membrane overnight on ice at 20 V. The membrane was blocked in 5% skimmed milk and probed with MSA180 primary antibodies (1:250) and HRP-conjugated secondary antibody (1:5000). The blots were developed using ECL reagent (Thermo Fisher).
Immunofluorescence assay
The slides for the late-stage schizonts were prepared by smearing infected erythrocytes onto glass slides. The slides were then air-dried and fixed by dipping in pre-chilled methanol for 30 min. The fixed slides were blocked with 3% BSA (Sigma Aldrich) prepared in PBS overnight at 4 °C, and then incubated with antibodies against the PfMSA180 recombinant proteins (1:100) along with marker antibodies (MSP-1, Exp-1, 1:200 dilution prepared in PBS) for 2 h at room temperature. The slides were washed vigorously with PBS containing 0.05% Tween 20 (PBST) and PBS and incubated with the corresponding secondary antibodies labelled with Alexa Fluor 488 and 594 (Thermo Fisher Scientific) for 1 h. The slides were then washed again with PBST and PBS, mounted in ProLong Gold antifade reagent and DAPI (Invitrogen) and viewed by confocal microscopy (Olympus FluoView™ FV1000) and processed using Imaris and NIS element.
Immunoprecipitation
Synchronized P. falciparum 3D7 schizont stage parasites (44–48 h), were harvested by centrifugation at 2,000 g for 5 min. The erythrocytes were lysed by incubating the pellet in 0.15% saponin for 10 min on ice. Immunoprecipitation was performed using the Pierce Co-Immunoprecipitation kit. The parasite material was lysed on ice by adding an equal volume of cell lysis buffer and 2X Protease inhibitor cocktail (Roche) for 3 h. The lysate was collected after centrifugation at 15,000 g for 30 min at 4 °C40. The lysate was cleared by incubation with control agarose resin for 1 h at room temperature with a gentle end-to-end mixing. The pre-cleared lysate was collected following centrifugation at 1,000 g for 1 min. In the meantime, IgG from day 70 sera was bound and cross-linked to protein A/G agarose resin. 2 mg of the pre-cleared schizont lysate was incubated with antibodies crosslinked to the resins overnight at 4 °C. The column was washed and the immunoprecipitated samples were eluted with elution buffer. The samples were trypsin digested and analysed by mass spectrometry (V proteomics, New Delhi, India).
Generation of inducible gene knockout parasites
Identification of guide RNA (gRNA) sequence and insertion into CRISPR/Cas9 plasmids: The gRNA sequences were identified using bioinformatic tools (protospacer and CRISPR guide RNA/DNA design tool at EuPaGDT41). The identified gRNA sequences were procured from Sigma. The gRNA oligonucleotides were phosphorylated at 37 °C for 30 min using T4 polynucleotide kinase (NEB) and annealed by a temperature ramp down from 94 °C to 25 °C at 5 °C/min. The Cas9 vector (pDC2-Cas9-hDHFRyFCU)18 was digested at 37 °C with BbsI restriction enzyme. The digested vector was dephosphorylated with calf intestinal alkaline phosphatase (Roche) at 37 °C for 30 min followed by incubation at 50 °C for 30 min. The phosphatase was deactivated by adding EDTA and incubating at 75 °C. The digested vector was column purified (QIAquick PCR purification kit, Qiagen).
The annealed oligonucleotides were stored at 4 °C and then diluted with nuclease free water in a 1:200 ratio and ligated with the digested vector. The ligation mixture was transformed into XL10 gold competent cells and insertion of the gRNA into plasmids was confirmed by sequencing of miniprep plasmid DNA (Qiagen Miniprep kit). Midi plasmid preparation was performed for selected clones (Qiagen midi kit).
Design of repair plasmids (used by the parasites for homologous DNA repair after Cas9 double strand DNA cleavage): Since the pfmsa180 gene lacks introns, loxP sequences were inserted using the loxPint approach28. DNA sequence between the two loxP sites was recodonised and synthesized by GeneArt. The repair plasmids were transformed into XL10 gold competent cells and maxi plasmid preparations were prepared (Qiagen Maxi Kit). The repair plasmids were designed with a suitable restriction enzyme site at either end.
To create the plasmid mixture for transfection of P. falciparum II-3 schizonts, the plasmid was linearized by restriction enzyme digestion and mixed with Cas9-guide RNA plasmid at a molar ratio of 5:1, and then ethanol precipitated at − 20 °C overnight. The DNA precipitate was collected by centrifugation at 13,000 g for 30 min at 4 °C; the ethanol was decanted and the pellet air dried. The DNA was dissolved in 10 µl Tris–EDTA buffer and stored at − 20 °C.
Parasite culture and transfection: Schizont stages of the II-3 parasite were obtained from tightly synchronised cultures by percoll purification, and electroporated with the DNA mix (Amaxa 4D-Nucleofector using P3 Primary Cell Kit). Following transfection, the cultures were treated with 2.5 nM WR99210 for 4 days of selection and then cultured further without drug until viable parasites reappeared (10–14 days). Genomic DNA was isolated (Qiagen genomic DNA isolation kit) and analysed by PCR for integration. Marker-free parasite clones were generated seven weeks post transfection by limiting dilution cloning. These were tested by adding 10 nM rapamycin to early ring stage parasites to allow DiCre recombinase-mediated excision of DNA18. DNA was isolated from the rapamycin treated and control (DMSO-treated) samples (Qiagen) and excision was monitored by PCR.
Statistical analysis
The results were analysed using MS Excel and reported as mean ± Standard error post data preparation.
Ethics declarations
The animal experiments were approved by Jawaharlal Nehru University (JNU) Institutional Animal Ethics Committee (IAEC Code No: 09/2019) as described by the ARRIVE guidelines (PLoS Bio 8(6), e1000412,2010). The use of transgenic parasites was approved by JNU Institutional Biosafety Committee (IBSC No: JNU/IBSC/2020/52) as per the guidelines of Department of Biotechnology, Government of India. The transgenic parasites were generated at The Francis Crick Institute. All methods were performed in accordance with the relevant guidelines and regulations.
