4.3 Indirect immunofluorescence analysis
IFA was performed essentially according to 60, where infected RBCs (iRBCs) were settled onto a poly-L-lysine (Sigma, P8920) coated coverslip and fixed with 4% paraformaldehyde/0.0075% glutaraldehyde. Following fixation, the cells were permeabilised with 0.1 M glycine/0.1 % Triton X-100 for 12 minutes at room temperature (RT). Coverslips were probed with rabbit anti-Nluc IgG (12.5 µg/mL), mouse anti-EXP2 (10 µg/mL), rabbit anti-ERC (1:1000), mouse anti-FLAG M2 (Sigma, 10 µg/mL), mouse anti-HA (Sigma clone HA-7; 1:500). After washing, goat anti-rabbit Alexa Fluor 594 nm and goat anti-mouse Alexa Fluor 488 nm (1:2000) secondary antibodies (Invitrogen) were applied for 1 h at RT. Fixed material was mounted in VECTASHIELD with DAPI and imaged on Zeiss Cell Axio-Observer (Carl Zeiss). Image acquisition was performed with Zen Blue imaging software.
4.4 Quantification of Imaging Data
Image analysis was carried out according to42.
4.5 Chemical Cross-Linking of P. falciparum Culture and Immunoprecipitation
RBCs infected with the HSP101-HAglmS /PEXEL-Nluc-mDH-FL parasites were enriched through magnetic purification and their proteins were solubilised in 20x pellet volume of IP lysis buffer (1% Triton X-100, 0.1% SDS, 150 mM NaCl, 10 mM Tris-HCl pH 7.4) supplemented with cOmpleteTM Protease Inhibitor Cocktail (Roche) and subjected to 2 freeze and thaw cycles. The lysate was clarified by centrifugation and incubated overnight at 4oC with anti‐HA monoclonal agarose (Sigma-Aldrich). Following incubation, the agarose beads were washed 5x with 1 mL IP lysis buffer and the proteins were eluted with 50 µL 2x NRSB (100 mM Tris-HCl pH 6.8, 4 mM EDTA, 4% SDS, 0.01% bromophenol blue, 20% (v/v) glycerol). For Nluc IgG immunoprecipitations, the parasite lysates were incubated overnight at 4oC with 10 µg IgG-purified anti-Nluc antibody. Following incubation, protein A-Sepharose 4B (Invitrogen) was added to bind the immune complexes and samples were incubated for an additional 1 h at RT. The beads were washed, and proteins eluted as above. In all cases, both input and elution fractions were reduced in 200 mM DTT at 70oC for 5-10 mins prior to SDS-PAGE separation, western blot, and immunoblotting.
4.6 Western Blotting
Proteins transferred from the gels to a nitrocellulose membrane using iBlot® Blotting System (Invitrogen). The blots were blocked in 1% casein in PBS and probed with primary antibody (Table 2.3) diluted in the blocking buffer overnight at 4oC. The blots were washed and probed with goat anti-mouse or goat anti-rabbit fluorescence-based (Alexa Fluor 700 and 800 nm, Invitrogen) antibodies diluted 1/10,000 in blocking buffer for 1 h at room temperature followed by 3 washes with 1x PBS. The fluorescent secondary antibodies were visualized with a LI-COR Odyssey FC imaging system. Densitometry analysis was performed with Image Studio v. 1.0.
4.7 Biochemical PEXEL Cleavage Assays
The cleavage assay was performed as described in Hodder et al. (Hodder, Sleebs et al. 2015). 2 nM of P. vivax PMV in buffer (25 mM Tris-HCl pH 6.4 and 25 mM MES pH 6.4) was incubated with 5 μM FRET peptide substrates representing WT and mutant KAHRP, STEVOR and Hyp1 sequences (Supplementary Table 5) in a total volume of 20 μL. Samples were incubated at 20°C for 20 h and measurement was carried out using Envision plate (PerkinElmer) reader (ex. 340 nm; em. 490 nm). Biochemical P. vivax PMV inhibitory assays (20 μL total volume) were performed using 2 nM P. vivax PMV in buffer (25 mM Tris-HCl and 25 mM MES, pH 6.4) with 5 μM FRET KAHRP_WT fluorogenic peptide. Assay reactions were incubated at 37°C for 2 h in the presence of peptides (10 points dose-response, 1 in 2 dilution series starting at 100 nM) representing STEVOR and Hyp1 sequences (Table 2.2). Fluorescence was measured with an Envision plate (PerkinElmer) reader (ex. 340 nm; em. 490 nm). To determine the level of PMV inhibition, “nonlinear regression four-parameter to fit analysis” using Domatics software (version 5.3.1612.8630) was performed.
4.8 Protein Solubility Analysis
Protein solubility profiling was performed according to Gruring et al.38. 5 µL of parasite pellet (obtained through magnetic separation or saponin lysis) was resuspended in 100 µL hypotonic buffer (5 mM Tris-HCl pH 8.0) and subjected to 1 cycle of freeze and thawing. The soluble fraction was separated from the pellet by centrifugation at 16,000 x g for 5 minutes at 4oC. The pellet was then incubated sequentially with 100 µL 0.1M Na2CO3 pH 11.5 and 1% Triton-X in H2O for 30 minutes at 4oC. Soluble fractions from each incubation were transferred into a new tube. Soluble fractions were mixed with 20 µL 6x NRSB (300 mM Tris-HCl pH 6.8, 12 mM EDTA, 12% SDS, and 0.03% w/v bromophenol blue, 60% v/v glycerol) and the pellet was resuspended in 120 µL 1xNRSB. All samples were kept at -20oC until used.