4.10 Liquid chromatography/ Mass spectrometry (LC/MS)
To determine the cleavage sites of the processed products in rPM X, the indicated bands in figure 1A were excised from the gel and were subjected to LC/MS analysis. Briefly, the gel samples were destained, reduced and alkylated prior to trypsin digestion. Digest was aspirated and combined with further extraction of the gel piece with 60% acetonitrile (ACN) in 1% trifluoroacetic acid (TFA). The extracted peptides were dried down and each sample was resuspended in 10 µL 5% ACN/0.1% formic acid (FA). 5 µL was analyzed by LC-MS with a Dionex RSLCnano HPLC coupled to an Orbitrap Fusion Lumos (Thermo Scientific) mass spectrometer using a 2 h gradient. Peptides were resolved in a 75 µm x 50 cm PepMap C18 column (Thermo Scientific) with following gradient: Time = 0–4 min, 2% B isocratic; 4–8 min, 2–10% B; 8–83 min, 10–25% B; 83–97 min, 25–50% B; 97–105 min, 50–98%. Mobile phase consisted of A, 0.1% FA; mobile phase B, 0.1% FA in ACN. The instrument was operated in the data-dependent acquisition mode in which each MS1 scan was followed by Higher-energy collisional dissociation (HCD) of as many precursor ions in 2 second cycle (Top Speed method). The mass range for the MS1 done using the FTMS was 365 to 1800 m/z with resolving power set to 60,000 at 400 m/z and the automatic gain control (AGC) target set to 1,000,000 ions with a maximum fill time of 100 ms. The selected precursors were fragmented in the ion trap using an isolation window of 1.5 m/z, an AGC target value of 10,000 ions, a maximum fill time of 100 ms, a normalized collision energy of 35 and activation time of 30 ms. Dynamic exclusion was performed with a repeat count of 1, exclusion duration of 30 s, and a minimum MS ion count for triggering MS/MS set to 5000 counts.
Data Analysis
All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.5.1.0). Mascot was set up to search against provided sequence along with common contaminants. The digestion enzyme was set as semiTrypsin (to account for nontryptic termini). Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 10 ppm. Oxidation of methionine, carbamidomethylation of cysteine, and acetylation of N-terminal of protein were specified in Mascot as variable modifications. Scaffold (version Scaffold_4.8.2 Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established under 1% FDR by the Peptide Prophet algorithm35 with Scaffold delta-mass correction. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm36.
4.11 Size exclusion chromatography
For gel filtration, rPM X samples were pretreated in acidic PM X activation buffer (25 mM MES, 25 mM Tris, pH 5.5) for 30 min at room temperature. Before sample runs, the Superdex 200 10/300 column (GE Healthcare) was equilibrated in the same buffer. Following runs, the samples were collected as 0.5 ml fractions. Fractions of interest (as indicated in Fig. 4) were further concentrated using an Amicon ultra 3 kDa mol. wt. cutoff filter. Finally, samples were resolved by SDS-PAGE and the gel was stained with Coomassie dye. For gel filtration under denaturation, every step was done as mentioned except that the buffers were supplemented with 6M guanidine hydrochloride. For denaturation, sample was treated with 6M guanidine hydrochloride and heated at 60oC for 45 min before loading onto the size exclusion column.
4.12 Native gel electrophoresis
For native PAGE, rPM X was preincubated in buffers (25 mM MES and 25 mM Tris) with indicated pH conditions for 1 h at RT. Samples were then mixed with 5x native gel loading buffer (312.5 mM Tris, pH 6.8, 50% glycerol, 0.05% bromophenol blue) and run in a discontinuous gel with 4% and 10% polyacrylamide for the stacking and the resolving parts respectively. Sodium dodecyl sulphate (SDS) was excluded both from the gel and the running buffer (192 mM glycine, 25 mM Tris, pH 8.3). Gel was subsequently stained with Coomassie brilliant blue R250 (ThermoFisher) and destained in 30% methanol, 10% glacial acetic acid.
4.13 Western blots
For PM X knockdown western blots (Fig. 2b), following synchronization, each culture was split into two plates. To one plate, aTc was added, and to the other equal volume of vehicle (DMSO) was added. 46 h post invasion, schizonts were collected by saponin lysis and stored at -800C until further processing. Samples were subsequently lysed in RIPA buffer supplemented with protease inhibitor cocktail and hemozoin was removed by centrifugation. Cell lysates were mixed with 4x sample loading buffer and heated at 990C for 5 min. Fractions of equal cell equivalents were subjected to SDS-PAGE and immunoblotting.
For processing inhibition assays (Fig. 7), PM Xapt parasites were synchronized and grown up to 44 h of age, as above. The infected RBCs from + aTc were then separated by MACS and resuspended in a small volume supplemented with either E64d (10 µM) or with compound 1 (1.5 µM) for the next 6 h. Cultures were then separated from the culture supernatant and the pellet fractions were boiled with 2x sample loading buffer.
Primary antibodies included rabbit anti-AMA1 (1:500), rabbit anti-SUB1 (1:1000), rabbit anti-HA (1:1000), rabbit anti-Sera5 (1:1000), mouse anti PM V (1:500), rabbit anti-Rh5 (1:500), mouse anti-GFP (1:1000), mouse anti-his (1:1000), and rat anti-FLAG (1:1000). For all, appropriate IRDye conjugated secondary antibodies were used at 1:10000 dilution. Blots were visualized on an Odyssey imaging system (Licor).
4.14 Immunofluorescence assays
For IFAs, cells were fixed as described previously37. Briefly, synchronized and C1-treated mature schizonts were fixed in 3.7% paraformaldehyde for 15 min and blocked in 3% BSA in PBS overnight at 4oC before antibody staining. The antibodies used for IFA were: rabbit anti-HA (Invitrogen, 1:500), mouse anti-HA (1:500), mouse anti-GFP (Invitrogen;1:500), mouse anti-MSP1 (1:500), rabbit anti-AMA1 (1:500), mouse anti-RAP1 (1:500), rabbit anti-EBA175 (1:500), rabbit anti-RON4 (1:500) and rabbit anti-BiP (1:250). The secondary antibodies were used as 1:2000 dilutions and were conjugated to Alexa Fluor 488 or 546 (Life Technologies). Cells were mounted with ProLong and 4’,6’-diamidino-2-phenylindole (DAPI) (Invitrogen) and imaged using a Zeiss Imager M2 Plus wide field fluorescence microscope, using a 63x objective for epifluorescence imaging.
For confocal imaging, samples were analyzed with a Zeiss LSM880 laser scanning confocal microscope with Airyscan (Carl Zeiss Inc. Thornwood, NY). A Plan-Apochromat 63X (NA 1.4) DIC objective and ZEN black software (version 2.1 SP3) were used for image acquisition. The image analysis software Volocity (version 6.3) (PerkinElmer, Waltham, MA) was used for 3-dimensional rendering of Z slices acquired through the depth of the parasites. For SR-SIM microscopy, samples were analyzed with a Nikon SIM super resolution microscope using a 100x objective. Image processing, analysis, and display were performed using Axiovision or Zen Blue. Adjustments to brightness and contrast were made for display purposes.
4.15 Immunoelectron microscopy
Samples for immunoEM were prepared as before3. Briefly, the infected RBCs were fixed in 4% paraformaldehyde (Polysciences Inc., Warrington, PA) with 100mM PIPES/0.5mM MgCl2, pH 7.2 for 1 h at 4°C. Samples were then embedded in 10% gelatin and infiltrated overnight with 2.3M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl2 at 4°C. Samples were trimmed, frozen in liquid nitrogen, and sectioned with a Leica Ultracut UCT cryo ultramicrotome (Leica Microsystems Inc., Bannockburn, IL). 50 nm sections were blocked with 5% FBS/5% NGS for 30 min and subsequently incubated with primary antibodies for 1 h, followed by secondary antibodies conjugated to 12 nm or 18 nm colloidal gold (1:30) for 1 h. Sections were washed in PIPES buffer followed by a water rinse, and stained with 0.3% uranyl acetate/2% methyl cellulose and viewed on a JEOL 1200EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA). All labeling experiments were conducted in parallel with controls omitting the primary antibody, which were consistently negative at the concentration of colloidal gold-conjugated secondary antibodies used in these studies.
4.16 Statistical analysis
Unless specified otherwise, assay values in all figures are averaged from three independent repeats and error bars represent standard deviations. Differences were assessed by the Student’s t-test using the Microsoft Excel software. For quantification of colocalization in figure 5c, epifluorescence images were analyzed by ImageJ and a Pearson’s colocalization coefficient was determined for the indicated antigen pairs. P values indicating statistical significance were grouped in all figures (***: p < 0.001, **: p < 0.01, *: p < 0.05).