Parasite cultures

Plasmodium falciparum strains 3D7 and NF54 were obtained from BEI resources, Malaria Research and Reference Reagents Resource Centre (MR4), American Type Culture Collection (ATCC). Parasites were maintained in O⁺ human erythrocytes (5% hematocrit) in RPMI-1640 supplemented with 0.5% AlbumaxII and 50 μg/mL hypoxanthine (cRPMI1640). Cultures were maintained at 37 °C under a mixture of 5% CO₂, 3% O₂, and 91.8% N₂ or 5% CO₂. Fresh erythrocytes were used to dilute parasites with fresh culture media to maintain 3–5% parasitemia at 5% haematocrit⁴⁰. For culturing various transgenic lines, relevant drugs were used as described above. Parasite synchronization was carried out using sorbitol as described previously⁴¹.

Generation of plasmid constructs and transfection of parasites

PfPPM2-3xHA-GlmS^3D7/NF54

In order to fuse PfPPM2 with glmS and introduce a 3xHA tag at the C-terminus, a homology region corresponding to its 3’-end PfPPM2 was PCR amplified using primers (1/2) from P. falciparum 3D7 genomic DNA and cloned in pGlmS-SLI vector (a kind gift from Prof. Alan Cowman and further modified by Dr. Rahul Rawat by incorporating 2 A skip-NeoR cassette) using sites for BglII and PstI. Subsequently, PfPPM2-3xHA-glmS construct was then transfected in 3D7 and NF54 parasites. For the transfection of this and other plasmids described below, 8–10% ring stage parasites were electroporated with ~100 μg plasmid DNA. Parasites were maintained initially on 2-10 nM WR99210 followed by 400 μg/ml of G418 for selection linked integration. A mixed population of parasites, which included both integrants and wild type parasites, was observed upon genotyping of drug-selected parasites. The mixed population was subjected to limited dilution cloning, the clones obtained after cloning were verified for correct 5’ and 3’ integration and the absence of the wild type unaltered region by genotyping PCR. PCR amplicons were sequenced for further confirmation. Genotyping was performed by using sets of PCR primers indicated in Supplementary Table 2.

PfPPM2-3xHA-GlmS^{NF54:HP1/HP1-S33A-Flag}

To generate pSLI-3x-Flag-yDHODH-BSD vector, first amplification of 2A-yDHODH fragment was done from pSLI-N-sandwich loxP vector⁴² using primer (7/8) and cloned using KpnI and XhoI sites in pSLI-HA-glmS-BSD (a kind gift from Dr. Asif Mohmmed and further modified by Dr. Rahul Rawat by incorporating 2 A skip-NeoR cassette) vector to yield pSLI-3x-Flag-2A-yDHODH-BSD, which resulted in the replacement of the 3xHA-tag by 3xFlag coding sequence and introduction of 2 A skip peptide along with yDHODH cassette and removal of NeoR and glmS sequence. For tagging HP1 at the C-terminal end with 3x Flag tag, 3’- homology region was amplified from 3D7 genomic DNA using primer (9/10) and cloned using SpeI and KpnI sites to generate pSLI-HP1-3x-Flag-yDHODH-BSD construct. For generating, S33A-mutant tagged with flag, PCR primers (11/10)-in which codons for S33 were replaced with those for A-were used to clone the homology region using SpeI and KpnI sites of pSLI-3x-Flag-yDHODH-BSD vector. This construct was transfected in PPM2-HA-glmS^NF54 parasite line and subsequently transgenic parasites were selected by using blasticidin (2.5 µg/ml) and recombinants were enriched using 1.5 μM DSM-1 and drug resistant parasites were genotyped for 5’-and 3’-integration and to check for the unmodified wild type locus of HP1. The desired mutation was confirmed by Sanger sequencing.

PfPPM2-HA-glmS^Nf54:HP1-GFP^OE or HP1/S33A/S33D-GFP-DD

HP1 was cloned in either pARL-GFP with BSD (a kind gift from Dr. Mohmmed Asif) or pARL-GFP-DD vector, which was modified as described below. For generating the pARL-HP1-GFP-DD, HP1 was amplified by PCR from 3D7 genomic DNA using primers (16/17) and GFP coding sequence was amplified from cpARL-GFP-BSD vector using primers (18/19) and the destabilization domain (DD) was amplified using primers (20/21) from pTEX-HA-DD vector. The HP1 and GFP domain was fused by overlapping PCR using primer (16/19) and GFP and DD domain was fused together using primer (18/21). The final overlapping PCR to obtain HP1-GFP-DD fused together was done by using primer set (16/21) and cloning was performed using KpnI and AvrII restriction sites of cpARL-GFP-BSD vector. S33A mutations in HP1 were generated by site directed mutagenesis using primers (22/23) and S33D mutation was generated using primers (24/25), which were confirmed by sequencing. Subsequently, mutants were cloned in pARL-GFP-DD vector like the WT HP1. For pARL-HP1-GFP construct, HP1 was cloned in pARL-GFP-BSD vector using primers 16/17’ using KpnI and AvrII sites.

These plasmid constructs were transfected in PPM2-HA-glmS^NF54 for generating PfPPM2-HA-glmS^Nf54:HP1-GFP^OE or NF54 parasites for HP1/S33A/S33D-GFP-DD parasites and were selected with 2.5 μg/ml of Blasticidin.

Growth rate assays

PfPPM2-HA-glmS parasites were cultured in the presence of 10 nM WR99210 and 400 μg/ml G418 and PfPPM2-HA-glmS^Nf54:HP1-GFP^OE parasites were cultured in the presence of 10 nM WR99210 (Jacobus Pharmaceuticals), 400 μg/ml G418 (TM Media; 3341) and 2.5 μg/ml of Blasticidin (Sigma-Aldrich;15205). Subsequently, ring stage parasites were seeded at ~0.5% parasitemia at 2% hematocrit after synchronisation. For the depletion of PfPPM2, ring stage parasites were typically treated with 2.5 mM glucosamine (GlcN) (Sigma-Aldrich; G1514) for one cycle and parasite growth was assessed at an interval of 24-h for additional 144-h. For growth rate assays with HP1/S33A/S33D-GFP-DD lines, parasites were synchronized and seeded at ~0.5% ring parasitemia and parasites were cultured in the presence or absence of Shield-1 (250 nM) (Cheminpharma, LLC, USA) for two cycles. Typically, samples were collected after every 24 h for making thin blood smears as well as FACS analysis.

Giemsa-stained thin blood smears were microscopically examined and various parasite stages (rings, trophozoite, schizonts) were counted to determine the parasitemia. For determining parasitemia using flow cytometry, samples were fixed with 1% PFA and 0.0075% glutaraldehyde solution and kept on an end-to-end rocker for 15 min. After completion, samples were either stored at 4^oC or processed directly for Hoechst 33342 staining for 10-min at 37°C. Samples were then washed at least twice with FACS buffer followed by analysis on BDverse (BD biosciences) for 10,000–100,000 events per sample⁴³. The data was processed and analyzed using FlowJo V.10.10.0 software.

Assessment of parasite division

Parasite lines were synchronized and GlcN treatment was provided for one cycle, E64 (10 µM) (Sigma-Aldrich; E3132) was added at ~40 h.p.i. (cycle 1) schizonts for 4–5 hours. Thin blood smears were prepared, stained with Giemsa images of mature schizonts were captured on a Leica bright field microscope. The numbers of merozoites from at least 50 schizonts for each condition, per replicate, were counted and average number of merozoites per schizont was determined.

Gametocyte conversion assay

For inducing gametocytogenesis, sexual conversion was induced as previously described with some minor modifications^31,44. Briefly, PPM2-HA-glmS^NF54 parasites were treated with 2.5 mM GlcN for one cycle (pre-treatment). Subsequently, parasites were seeded at 2% rings in 5% haematocrit. In the next cycle, when parasitemia reached ~10%, gametocytogenesis was induced by replacing serum containing complete RPMI-1640 (cRPMI) medium with serum free medium containing Fatty acid free BSA (Sigma Aldrich; A6003), Oleic acid (Sigma Aldrich; O1008), Palmitic acid (Sigma Aldrich; P0500). After ~24-h, serum free medium was removed followed by supplementation of cRPMI and treatment with 50mM N-acetyl-D-Glucosamine (Sigma-Aldrich; A8625) was provided for 5 days to eliminate the asexual parasites unless indicated otherwise. Giemsa smears were made periodically and the number of gametocytes was counted at day 5 p.g.i. Sexual conversion rates were determined by counting gametocytes formed at day 5 relative to the initial parasites, which were mainly asexual rings^45,46. In order to assess the sexual conversion of the HP1/S33A/S33D-GFP-DD parasites, sorbitol synchronization was performed at the ring stage and parasites were left untreated or treated with Shld-1 (250 nM). Subsequently, IFAs were performed after 4–6 days on blood smears using anti-GFP (Roche; SKU-11814460001) and anti-Pfs16 (MRA-1276) or anti-Pfs230 (MRA-878A) antibodies. The number of Pfs16/Pfs230-stained parasites that represented gametocytes was counted. Since pS33-HP1 antibody (Antagene) was raised in rabbits, co-staining was done using anti-Pfs230 which was raised in mice.

Parasite invasion assay

Invasion assays were carried out as described previously with slight modifications^43,47. Following sorbitol treatment, parasites were cultured in the presence or absence of glucosamine (2.5 mM GlcN) for one cycle. After 90-h (cycle 1, schizonts), MACS columns (Miltenyi Biotec) were used to purify schizonts, which were washed twice and resuspended in RPMI 1640 medium with fresh RBCs to achieve 2% hematocrit. Invasion experiments were performed using 0.5–1% schizont / 2% hematocrit in a 6-well plate in a gas chamber equilibrated with gas mixture described above at 37°C along with shaking at 80 rpm and samples were collected every 2-h. Flow cytometry was used to determine the number of schizonts and rings for which parasites were fixed using 1% PFA + 0.0075% glutaraldehyde in FACS solution (0.3% BSA + 0.02% Sodium azide in 1xPBS solution) followed by staining with Hoechst 33342 (1:10,000 dilution) which was prepared in FACS solution. Flow cytometry data was acquired using BD FACS verse and analysed using FlowJo V.10.10.0 software (Tree Star, Ashland, OR).

Immunofluorescence Assay (IFAs)

Immunofluorescence assays (IFA) were performed on thin blood smears as previously described⁴⁸. Briefly, air-dried thin blood smears were fixed with cold methanol and acetone mix (1:1, v:v) for 2-min followed by blocking with 3% BSA for 45-min at room temperature. Subsequently, smears were incubated with primary antibodies for 12-h at 4 ^°C. After washing with blocking buffer, Alexa fluor mouse/rabbit 488/594-labelled secondary antibodies were added (Invitrogen) for 2-h at ambient temperature. Finally, Vecta shield mounting media was used (Vector Laboratories Inc.), which contained DAPI to label nuclei. Fluorescence microscopy was performed using Axio Imager Z1 microscope or a LSM980 confocal microscope (Carl Zeiss). The images were processed using AxioVision 4.8.2 or Zeiss ZEN blue 3.1 or 3.9 software and best representative z-stacks were used for illustrations in the figures unless indicated otherwise.

Ultrastructure-Expansion Microscopy

PfPPM2-HA-glmS^Nf54 parasite lines were synchronized and GlcN treatment was provided for one cycle. Further, at ~92–94 h.p.i. (cycle 1) schizonts were fixed using 4% para-formaldehyde (PFA) in PBS at 37°C for 20-min. Sample preparation of P. falciparum parasites for U-ExM was performed as previously described with slight modifications^12,49. Immuno-labelling was performed using primary antibodies against α-tubulin (1:500 dilution, SAB3501072, Sigma) anti-centrin antibody (1:200 dilution, Sigma 20H5), anti-HA antibody (1:200 dilution, 12CA5 Roche), anti-PfGAP45 antibody (1:500 dilution, generated previously by Sharma laboratory) and sytox (S11830, Invitrogen). Secondary antibodies anti-mouse Alexa 568 (A21134, Invitrogen), anti-rabbit Alexa 488 (A27034, Invitrogen) and were used at dilutions 1:1000. Images were acquired using a LSM980 confocal microscope (Carl Zeiss) and images were processed using Zeiss ZEN blue 3.1 or 3-.9 software. Maximum Intensity Projection (MIP) was provided for all U-ExM images. The laser power was maintained constant for imaging control and treated samples unless indicated otherwise.

Immunoblotting

Parasite cultures were collected and iRBCs were lysed using 0.05% saponin (w/v) followed by incubation on ice for 10-min. Centrifugation at 8000 rpm was done to isolate the parasite pellet, which was washed three times with pre-chilled PBS. The parasite pellet was re-suspended in lysis buffer (10 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, and complete protease inhibitor cocktail; Roche Applied Science) or 2% SDS and then homogenized by either passing the solution through a 26-gauge needle or by using sonication at 60% amplitude for 20-sec (1-sec ON/OFF). The supernatant from the centrifugation of the lysates at 14,000 g for 30-min at 4 °C was used to estimate the amount of protein using a BCA protein estimation kit (Pierce). Nuclear protein lysates were prepared as described below.

After SDS-PAGE, proteins were transferred to nitrocellulose membrane and membranes with transferred proteins were blocked with 3% BSA/0.2% Tween 20 in 1xTBS (v/v) for 1-h. Primary antibody solutions were made in 3% BSA, diluted to the required concentration and membranes were incubated for 12-h at 4 °C. The membrane was then rinsed three times with 1xTBS/0.1% Tween 20 (TBS-T) and incubated with Horseradish Peroxidase (HRP) conjugated secondary antibody prepared in 3% skimmed milk/1xTBS-T or 3%BSA/1x TBS-T (used for phospho antibody-blots) for 2-h. The nitrocellulose membrane was once again washed with 1xTBS-T and detection was performed using West Pico or Femto chemiluminescence substrate from Pierce (USA) after exposure of the membrane to X-ray films.

Immunoprecipitation

Immunoprecipitation was performed on nuclear lysates. For making the nuclear lysate, fractionation was performed as previously described^28,29 with some minor modifications. Saponin-lysed infected red blood cells were lysed in ice-cold cell lysis buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitors) for 5-min. After a 5-min centrifugation at 5200 rpm (2500xg), the cytosolic extract was collected and stored at -80°C and the nuclei obtained were treated with DNase I and RNase A [(20 mM Hepes pH 7.4, 10 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 0.1% NP-40, 1x protease inhibitor (Roche Diagnostics), 1x PhosSTOP (Roche Diagnostics), 0.5U/µl DNase I, 1 µg RNase A] and incubated in 250 µl reaction volume for 5-min at 37°C). Reactions were adjusted to 500 µl using 2x high salt extraction buffer (20 mM Hepes pH 7.4, 1 M NaCl, 6 mM EDTA, 2 mM EGTA, 2 mM TCEP, 1x protease inhibitor (Roche Diagnostics), 1x PhosSTOP (Roche Diagnostics), 50% Glycerol, 0.1%NP-40) and nuclear proteins were extracted in 2.5 pellet volume of high salt extraction buffer (20 mM Hepes pH 7.4, 500 mM NaCl, 3 mM EDTA, 1 mM EGTA, 1 mM TCEP, 1x protease inhibitor (Roche Diagnostics), 1x PhosSTOP (Roche Diagnostics), 25% Glycerol, 0.1%NP-40) by vortexing for 30-min at 4˚C. After centrifugation at 20,800 g at 4°C for 30-min, supernatant was separated immediately and used for IP experiments after dilution. High salt nuclear extracts were diluted to 250 mM NaCl using dilution buffer (20 mM Hepes pH 7.4, 1 mM EDTA, 1 mM TCEP, 1x protease inhibitor (Roche Diagnostics), 1x PhosSTOP (Roche Diagnostics), 25% Glycerol).

For immunoprecipitation, ~200 µg of nuclear protein lysate was typically used and incubated with relevant antibodies at 4°C for 12-h followed by incubation with protein A + G sepharose beads for 5-h. After 3x washes with wash buffer, beads were boiled at 95˚C for 10-min. After boiling, supernatant was used for immunoblotting along with the input. After electrophoresis SDS-PAGE, lysate proteins were transferred to a nitrocellulose membrane and immunoblotting was performed as described previously^50,51 using specific antibodies and blots were developed using SuperSignal® West Pico or Femto chemiluminescence Substrate (Thermo Scientific) by following manufacturer’s instructions.

Generation of pS33-HP1 antibody and anti-HP1 antisera

Custom antibodies were raised against HP1 phosphorylated at S33 (anti-pS33-PfHP1) or an unphosphorylated version of the S33 (HP1 non-phospho) by M/s Antagene Inc. For phospho antibody generation, a peptide with a sequence: Cys-YLVKWKGY(p)SDDENTW and for non-phospho peptide Cys-YLVKWKGYSDDENTW was used for immunization. Phospho-antibodies were purified by affinity chromatography using these peptides and validation was done by the manufacturer by performing ELISA. For Western blotting, 1:100-200 dilution of the purified phospho and non-phospho antibody was used and for IFAs 1:25 of anti-pS33HP1 was used. Total HP1 antisera were raised in rabbit against bacterially expressed HP1 was a gift from Dr. Krishanpal Karmodiya, IISER Pune or was also raised against the same recombinant protein by immunizing rabbits. The approval from Institute Animal Ethics Committee of NII was taken for this purpose and recommended protocol and ethical regulations were followed.

SUrfaceSEnsing of Translation (SUnSET) assay or Puromycin incorporation assay

Protein synthesis was assessed using a SUnSET assay^21,52 for which parasites were treated with 1 µM puromycin (structural analog of tyrosyl-tRNA) (Sigma Aldrich; P8833) for 1-h. Parasites were harvested at trophozoite and schizont stages and protein lysates were prepared using 2% SDS, which were subjected to Western blot analysis. To ensure equal loading of proteins, the nitrocellulose membrane was stained with Ponceau S before blocking with 3% BSA for 1-h. After blocking, blots were incubated with anti-puromycin antibody (Merck; MABE343) overnight at 4°C. Subsequently, blots were washed with 1xTBST and incubated with α-FCY-subclass 2a mouse IgG antibody for 1-h. The blots were developed by Pico or Femto chemiluminescence substrate from Pierce (USA) after exposure of the membrane to X-ray films.

Quantitative RT-PCR

RNA isolation was carried out using TRIzol reagent (G Biosciences). Real time PCR was carried out using CFX96 Real Time PCR detection system (Biorad). Quantification of the expression was done by determining SYBR green incorporation in the amplicons (G Biosciences). For qRT-PCR, 1 μg of DNase free RNA was used for cDNA synthesis using Verso cDNA synthesis kit (Thermo scientific), as per the manufacturer’s recommendation. Random hexamers supplied with the kit were used for the cDNA synthesis. 18srRNA or Actin were used as a housekeeping gene for normalization. A typical PCR reaction conditions were: 95 °C for 3-min, 95 °C for 15-sec, 60 °C for 1-min and 40 cycles. Finally, the relative fold change in gene expression was determined as 2^-ΔΔCt. Primers details for the qRT-PCR are given in (Supplementary Table 2).

RNA sequencing

Nf54 (control) and PfPPM2-HA-glmS^Nf54 lines were treated with GlcN and were harvested at first cycle at the schizont stage (42-44 h.p.i) in triplicate. RNA isolation was performed using the phenol-chloroform extraction method, followed by quantification using a Qubit fluorometer (Thermo Fisher Scientific). For all samples, 1 μg of RNA was used as an input for mRNA enrichment using the NEBNext Poly (A) mRNA magnetic isolation module (New England Biolabs #E7490S) according to the manufacturer’s instructions. A NEBNext Ultra II RNA library prep kit for Illumina (New England Biolabs #E7770S) was used to prepare the library according to the manufacturer’s instructions, and quality checks were performed using Agilent TapeStation. Paired-end sequencing was performed on an Illumina Nextseq550 platform (75 bp read length).

RNAseq Data Analysis

RNA-seq data analysis was performed as described below. The quality of sequenced samples was accessed using FASTQC and adapters were trimmed with Trim Galore (https://doi.org/10.5281/zenodo.5127899) and cutadapt (https://doi.org/10.14806/ej.17.1.200). High quality trimmed reads were aligned in paired-end mode to Plasmodium falciparum 3D7 genome ver. 66 using HISAT2⁵³ with default settings. Differential gene expression analysis was performed using DEseq2⁵⁴ using standard analysis protocol. Gene set enrichment analysis was performed using ClusterProfiler²⁶ and results were plotted with custom R and python scripts.

Chromatin Immunoprecipitation (ChIP) and ChIP-qPCR

Parasites cultured in the presence (2.5 mM GlcN) or absence of glucosamine (GlcN) and were harvested at the end of first cycle at ~90 h.p.i by fixing the cells. The iRBCs were cross linked using 1% formaldehyde (Thermo Scientific, 28908) for 10-min. Subsequently, 150 mM glycine was then added for quenching the cross-linking reaction for 10-min at ambient temperature followed by centrifugation at 6000 rpm at 4˚C for 5-min. Pellet was washed three times using 1xcold PBS (supplemented with protease inhibitors) before proceeding for lysis. Sample homogenization was performed using swelling buffer (25 mM Tris pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP 40, 1 mM DTT, 0.5 mM PMSF, 1 × PIC) followed by cell lysis in sonication buffer (10 mM Tris–HCl pH 7.5, 200 mM NaCl, 1% SDS, 4% NP-40,1 mM PMSF, 1X protease inhibitor cocktail). First, pre-clearing was performed for 2-h at 4 °C by incubating the chromatin fraction with- protein A conjugated sepharose beads (G-Biosciences) with continuous gentle mixing. Pre-cleared lysate was incubated with the relevant antibody for 12-h at 4 °C followed by incubation with saturated Protein A Sepharose beads for 4-h at 4 °C. Bound chromatin was finally washed with low salt wash buffer, high salt wash buffer, LiCl wash buffer, TE wash buffer and eluted using ChIP elution buffer (1% SDS, 0.1 M sodium bicarbonate). Both IP sample and input were reverse cross-linked using 0.3 M NaCl overnight at 65 °C along with RNase followed by Proteinase K treatment at 42 °C for 1-h. Finally, bound DNA was purified using phenol-chloroform precipitation. The occupancy of genes of interest was assessed by ChIP-qPCR using the SYBR Green Master Mix (G Biosciences). The amount of template used for qPCR was 1 μl of 1% input DNA and 2 μl of undiluted ChIP DNA. qPCR was performed using previously described primers corresponding to AP2-G and Pfs16 locus (Supplementary Table 2)²⁷. The amount of DNA amplified was compared to the input and % recovery of bound DNA was determined.

Phosphoproteomics and Mass spectrometry

Proteomics Studies

Cell lysis and protein extraction and TMT labeling

The 3D7 or PfPPM2-HA-glmS^3D7 parasites were cultured and synchronized using sorbitol. Ring stage parasites were treated with glucosamine for 72-h. The parasites were subsequently harvested upon maturation to schizonts in cycle 1 ( ~ 44 hours post-infection), by lysing the infected red blood cells with saponin. The parasite pellet was washed and dissolved in protein extraction buffer (50 mM triethylammonium bicarbonate (TEAB, Sigma Aldrich; T7408), 2% SDS, and 1X protease and phosphatase inhibitors) followed by sonication and centrifugation. Protein estimation was done by Bicinchoninic Acid assay (BCA estimation kit, Sigma Aldrich; 23225). Equal amounts of protein were taken from all sets of parasites and subjected to reduction with 20 mM Dithiothreitol (DTT, Sigma Aldrich; D9779) at 60 °C for 30-min, followed by alkylation with 20 mM Iodoacetamide (IAA, Sigma Aldrich; I1149) for 10-min in the dark at ambient temperature. Subsequently, proteins were digested using trypsin and Lys-C mix (Promega, Madison, USA; V5073) used at a ratio of 100:1. Tryptic peptides were cleaned using a C18 column and dried with a SpeedVac concentrator (Thermo Fisher Scientific, USA). The peptides were quantified using the Pierce Quantitative Colorimetric Peptide Assay Kit (Sigma Aldrich; 23275). Further, 500 µg of peptides from each sample were processed for TMT labelling following manufacturer’s instructions (Thermo Scientific; 90110). Labelling efficiency of >95% was achieved and the reaction was stopped by adding 8 µl of hydroxylamine. The labelled peptides were pooled, dried, and cleaned using C18 Sep-Pak cartridges (Waters, Milford, MA, USA; WAT054955).

Phosphopeptide enrichment and fractionation

50 µg of the peptide sample was used for total proteome analysis, and the remaining sample was subjected to phosphopeptide enrichment. For the initial phosphopeptide enrichment, the TiO2 Phosphopeptide Enrichment Kit was utilized (Thermo Scientific; A32993). The subsequent enrichment step was performed on the collected flow-through using the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific; A32992). The eluents from both enrichment steps were combined and subjected to fractionation using C18 Stage Tips. A total of 24 fractions were collected and then concatenated into six fractions. The peptide samples were dried and dissolved in 0.1% formic acid (Thermo Scientific; 94318) prior to mass spectrometry analysis.

LC-MS/MS analysis by Data-Dependent Acquisition

Mass spectrometry analysis was performed on an Orbitrap Fusion Tribrid Mass Spectrometer linked to an Easy-nLC 1200 nano-flow UPLC system (Thermo Fisher Scientific, Germany). The peptide fractions were loaded on nanoViper column (2 cm, 3 µm C18 Aq) (Thermo Fisher Scientific) and were then resolved on an analytical column (75 µm × 15 cm, C18, 2 µm particle size) (Thermo Fisher Scientific). The peptides were separated by passing solvent A (0.1% formic acid) and solvent B (80% acetonitrile in 0.1% formic acid) using a gradient mode over a 120-min at a flow rate of 300 nl/min.

Data-dependent acquisition (DDA) mode was used for data acquisition. Precursor ions were acquired in full MS scan mode in the range of 400-1600 m/z, using an Orbitrap mass analyzer resolution of 120,000 at 200 m/z. An automatic gain control (AGC) target value of 2e5 with an injection time of 55 ms and dynamic exclusion of 30 seconds was used. The selection of the most intense precursor ions was performed at top speed DDA mode, selected using a quadrupole with an isolation window of 2 m/z. The filtered precursor ions were subsequently fragmented using higher-energy collision-induced dissociation (HCD) with 32 ± 3% normalized collision energy and MS/MS scans in the range of 110-2000 m/z were acquired by using Orbitrap mass analyzer with 30,000 mass resolution at 200 m/z. AGC target was set to 1e5 with an injection time of 200 ms. For each fraction, MS/MS data was acquired in duplicates.

Data analysis

MS/MS raw data was used to search against a combined protein database of P. falciparum 3D7 (downloaded from PlasmoDB web resource, version 46) and Homo sapiens (RefSeq v92) using SEQUEST and Mascot (version 2.4.1) via Proteome Discoverer v2.2 (Thermo Fisher Scientific) as described previously⁴⁷. A fold change cut-off of 1.5 with a p value ≤ 0.05 was used to identify significantly altered phosphorylated proteins The phosphorylation fold changes were normalized with respect to the total proteome data as reported earlier^47,55. Phosphosites that were identified differentially phosphorylated in two or more biological replicates were considered for the further analysis. The ptmRS node was used during the search to determine the probability of phosphorylation site localization and a ptmRS score ≥99% was used as a cut off. These proteins were then analyzed for the enrichment of biological processes using Gene Ontology (GO) analysis through PlasmoDB.

Densitometry and statistical analysis

Image J (NIH) software v1.54 was used to perform densitometry of Western blots. The band intensity of the loading control was used for normalization. Statistical analysis was performed using Graph Pad Prism v5 or 9 (Graph Pad software Inc USA). Data is represented as mean ± Standard error of mean (SEM), unless indicated otherwise and p < 0.05 was considered as statistically significant. No data was excluded from the analysis.

Reporting summary

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