Malaria kills over half a million people every year worldwide. A single-celled parasite called Plasmodium falciparum is responsible for the most lethal form of the disease. This malaria-causing agent is carried by mosquitos which transmit the parasite to humans through their bite. Once in the bloodstream, the parasite enters red blood cells and starts to replicate so it can go on to infect other cells.

Like our cells, P. falciparum is surrounded by a membrane, and further membranes surround a number of its internal compartments. To make these protective coats, the parasite has to gather a nutrient called choline to form an important building block in the membrane.

The parasite gets most of its choline by absorbing and digesting a molecule known as lysoPC found in the bloodstream of its host. However, it was unclear precisely how the parasite achieves this. To address this question, Ramaprasad, Burda et al. used genetic and metabolomic approaches to study how P. falciparum breaks down lysoPC.

The experiments found that mutant parasites that are unable to make an enzyme called GDPD were able to infect red blood cells, but failed to grow properly once inside the cells. The mutant parasites took up less choline and, as a result, also made fewer membrane building blocks. The team were able to rescue the mutant parasites by supplying them with large quantities of choline, which allowed them to resume growing. Taken together, the findings of Ramaprasad, Burda et al. suggest that P. falciparum uses GDPD to extract choline from lysoPC when it is living in red blood cells.

More and more P. falciparum parasites are becoming resistant to many of the drugs currently being used to treat malaria. One solution is to develop new therapies that target different molecules in the parasite. Since it performs such a vital role, GDPD may have the potential to be a future drug target.

The malaria parasite replicates within red blood cells (RBC). During its massive intraerythrocytic growth, the parasite produces de novo large amounts of membrane to support expansion of its parasite plasma membrane (PPM), the parasitophorous vacuole membrane (PVM) and other membranous structures of the growing parasite, as well as for formation of daughter merozoites. This extensive membrane neogenesis, which culminates in a sixfold increase in the phospholipid content of the infected RBC (Wein et al., 2018), requires an intense phase of phospholipid synthesis during the metabolically active trophozoite and schizont stages of the parasite life cycle.

Phosphatidylcholine (PC) is the most abundant membrane lipid in the malaria parasite, comprising 30–40% of total phospholipid (Botté et al., 2013; Gulati et al., 2015), and the parasite has evolved to produce this vital phospholipid via multiple enzymatic pathways from a variety of metabolic precursors from the host milieu (Wein et al., 2018; Kilian et al., 2018; Figure 1). Under normal conditions, ∼89% of PC is synthesized from free choline and fatty acids via the CDP-choline-dependent Kennedy pathway that is common among eukaryotes (Brancucci et al., 2017; Wein et al., 2018). Choline is phosphorylated to phosphocholine (P-Cho) by choline kinase (CK) (Ancelin and Vial, 1986b), then converted to CDP-choline by a CTP:phosphocholine cytidyltransferase (CCT) (Ancelin and Vial, 1989) and finally condensed with diacylglycerol (DAG) by a choline/ethanolamine-phosphotransferase (CEPT) (Vial et al., 1984) to produce PC. PC is also generated via an alternate serine-decarboxylase-phosphoethanolamine-methyltransferase (SDPM) pathway also found in plants and nematodes, that uses host serine and ethanolamine (Eth) as precursors (Pessi et al., 2004). In this case, precursor P-Cho for the CDP-choline pathway is produced by triple methylation of phosphoethanolamine (P-Eth) by a phosphoethanolamine methyltransferase (PMT) (Witola et al., 2008). Phosphoethanolamine is in turn generated from ethanolamine sourced either from the serum or converted from serine by an unidentified parasite serine decarboxylase. Unlike yeast and mammals, the malaria parasite is unable to convert PE directly to PC through phospholipid methyl transferase activity (Witola et al., 2008), so the Kennedy and SDPM pathways intersect only at the point of PMT activity (Figure 1). PC can also be potentially produced by direct acylation of lysoPC via the Lands’ cycle by an unknown lysophosphatidylcholine acyltransferase (LPCAT). However, this pathway is considered to not contribute significantly to PC synthesis under normal conditions (Wein et al., 2018).

Figure 1

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The crucial nature of PC biosynthesis for parasite survival has generated interest in this process as a potential target for antimalarial drug development (Ancelin et al., 1985; Ancelin and Vial, 1986a). Choline analogs have potent antimalarial activity (Ancelin et al., 2003), whilst inhibiting or disrupting enzymes in the PC synthesis pathways severely reduces or blocks intraerythrocytic growth. As examples of this, compounds that inhibit P. falciparum CK (PfCK) (Serrán-Aguilera et al., 2016) or PfCCT (Contet et al., 2015) in the CDP-choline pathway kill the parasite, and disruption of the PfPMT gene to block PC synthesis via the SDPM pathway results in morphological and growth defects but is not lethal (Witola et al., 2008). These findings suggest that the CDP-choline pathway provides the major route to PC synthesis whilst the SDPM pathway forms an important alternative route. An improved understanding of PC biosynthesis in Plasmodium may identify critical enzymes in the process that are potential drug targets.

The choline required for PC synthesis is primarily scavenged from host serum. Whilst free choline can cross the PPM efficiently through an unidentified carrier, choline transfer from serum into the infected RBC across the erythrocyte membrane via a parasite-induced new permeability pathway (NPP) appears rate-limiting (Ancelin and Vial, 1989; Biagini et al., 2004). Perhaps to overcome this limitation, the parasite has evolved to scavenge most of the required choline from exogenous lysoPC (Brancucci et al., 2017; Wein et al., 2018). Intriguingly, lysoPC also acts as an environmental sensor that controls sexual differentiation in P. falciparum. Active lysoPC metabolism into PC via the CDP-choline pathway prevents sexual commitment, while in contrast limited availability of lysoPC reduces formation of asexual progeny and triggers differentiation into the transmissible gametocyte stages (Brancucci et al., 2017). Metabolic labelling experiments showed that ∼68% of free choline in the parasite comes from exogenous lysoPC, indicating that the majority of the lysoPC is broken down to choline before entering PC synthesis (Brancucci et al., 2017). However, it is unclear how and where lysoPC is converted to choline in the parasite and the enzymes involved in this process are unknown.

LysoPC breakdown to free choline requires a two-step hydrolysis reaction: deacylation of lysoPC by a putative lysophospholipase to give glycerophosphocholine (GPC) that is then hydrolysed by a glycerophosphodiester phosphodiesterase (GDPD) to generate choline and glycerol-3-phosphate (G3P) (Figure 1). GPC catabolism by GDPD has been shown to maintain a choline supply for CDP-choline-dependent PC biosynthesis in model eukaryotes (Fernández-Murray and McMaster, 2005; Morita et al., 2016; Stewart et al., 2012). Only one putative glycerophosphodiesterase gene (PfGDPD; PF3D7_1406300) has been identified in the malarial genome (Denloye et al., 2012). The 475-residue predicted protein product has an N-terminal secretory signal peptide and a glycerophosphodiester phosphodiesterase domain (amino acid residues 24–466; InterPro entry IPR030395), which likely adopts a triosephosphate isomerase (TIM) barrel alpha/beta fold (https://alphafold.ebi.ac.uk/entry/Q8IM31) (Jumper et al., 2021; Rao et al., 2006; Varadi et al., 2022). The protein shares homology with prokaryotic GDPDs and contains a characteristic small GDPD-specific insertion (residues 75–275) within the TIM barrel structure. Recombinant PfGDPD has been shown to have hydrolytic activity towards GPC and localization studies have suggested that the protein is present in the parasite digestive vacuole (in which breakdown of host haemoglobin takes place), as well as the cytoplasm and parasitophorous vacuole (PV) (Denloye et al., 2012). Repeated failed attempts to disrupt the PfGDPD gene (Denloye et al., 2012) and a genome-wide mutagenesis screen (Zhang et al., 2018) suggests its essentiality for asexual stage growth. However, its role in parasite phospholipid metabolism remains unknown.

Here, we used a conditional gene disruption approach combined with chemical complementation and metabolomic analysis to examine the essentiality and function of PfGDPD in asexual blood stages of P. falciparum. Our results show that PfGDPD catalyzes a catabolic reaction that is key for lysoPC incorporation and PC synthesis in the parasite.

Mature human RBCs are highly streamlined, terminally differentiated cells that lack a nucleus or other internal membranous organelles, with no protein synthesis machinery and limited metabolic capacity. Since erythrocytic growth of the malaria parasite requires extensive membrane biogenesis, de novo PC synthesis by the parasite is essential. Host serum lysoPC, well established as the main precursor for PC synthesis in the parasite (Brancucci et al., 2017; Wein et al., 2018), can enter the parasitized erythrocyte efficiently through rapid exchange (Dushianthan et al., 2019) after which it may be transported into the parasite by exported phospholipid transfer proteins (van Ooij et al., 2013). However, the enzymes involved in the generation of choline from lysoPC have been unknown. Here we have established PfGDPD as an essential player in this process.

Ablation of PfGDPD expression produced a phenotypic defect during trophozoite development as either a direct or general stress response to disrupted PC synthesis, similar to that observed upon inhibition of other enzymes involved in CDP-choline pathway (González-Bulnes et al., 2011; Serrán-Aguilera et al., 2016; Contet et al., 2015). Unsurprisingly for an enzyme playing a key role in membrane biogenesis, previous transcriptional studies have shown that PfGDPD is expressed early in the erythrocytic cycle. Our observation that DiCre-mediated disruption of PfGDPD results in parasites that undergo normal development in the erythrocytic cycle of RAP-treatment (cycle 0) is therefore likely due to the presence of residual enzyme produced early during ring stage development (prior to gene excision) and persisting throughout that cycle. In support of this, PfGDPD-null parasites that were rendered completely devoid of the enzyme through propagation for several cycles in choline-supplemented media exhibited maturation defects within ∼24 hr of choline removal.

We infer that PfGDPD acts as a glycerophosphocholine phosphodiesterase and releases choline from the intermediary GPC during lysoPC breakdown from the following findings. First, site-directed mutagenesis showed that PfGDPD retains the same essential catalytic sites and metal-ion dependency as a bacterial choline-releasing GDPD enzyme (Shi et al., 2008). Second, supraphysiological concentrations of choline rescued the PfGDPD-null development and growth defect. Third, PC levels and incorporation of choline from lysoPC into PC are reduced in PfGDPD-null parasites. And fourth, PfGDPD affinity-purified from parasite lysate released choline from GPC in vitro.

Previous work has shown that supplying an excess of choline can rescue the growth of parasites in lysoPC-deprived conditions (Witola et al., 2008; Wein et al., 2018). High concentrations of exogenous choline partially surpass the bottleneck in choline transport across the red blood cell membrane (Biagini et al., 2004) and increase choline influx through infected RBCs (Kirk et al., 1991). LysoPC-deprived parasites can readily take up this choline and use it for PC synthesis as shown by previous metabolic labelling studies (Brancucci et al., 2017). The impact of exogenous choline was well pronounced in our PfGDPD-null mutants as they completely failed to survive in the absence of choline but achieved near-wild type growth rates in high choline concentrations. This complete dependence on exogenous choline despite the abundant presence of lysoPC (amounting to ∼17% of total lipid content Garcia-Gonzalo and Izpisúa Belmonte, 2008) in the Albumax II-supplemented culture media used in our studies or supplementation with GPC, is a further confirmation that ablating PfGDPD function interferes with choline acquisition from lysoPC and mimics a lysoPC-deprived state.

As well as its effects on PC levels, ablation of PfGDPD reduced both PE and PS content in the parasite, likely due to redirection of ethanolamine and serine precursors into PC synthesis. Consistent with this, ethanolamine supplementation marginally improved growth of PfGDPD-null parasites. Loss of PfGDPD also reduced – but did not eliminate – incorporation of lysoPC-derived choline. This suggests that alternate pathways such as direct lysoPC acylation into PC via the Lands’ cycle, two acylation steps to convert GPC to PC or the SDPM pathway may contribute to PC synthesis under choline-starved conditions. However, our results strongly suggest that PfGDPD-mediated choline release from lysoPC remains the primary, indispensable pathway to meet the choline requirements of the intraerythrocytic parasite.

The observed depletion of TAG in choline-starved PfGDPD-null parasites was unexpected since there is no previously reported role for a glycerophosphodiesterase in neutral lipid metabolism. An intense phase of de novo TAG biosynthesis from DAG is known to accompany trophozoite development (Palacpac et al., 2004; Vielemeyer et al., 2004), followed by a rapid hydrolysis of TAG in mature schizonts resulting in localized release of fatty acids essential for merozoite maturation prior to egress (Gulati et al., 2015). Our results could simply reflect the different developmental stages of the choline starved G1 and B4 parasites at the point of lipid extraction (Figure 5—figure supplement 3) as a result of the developmental arrest that inevitably occurs in G1 parasites upon choline deprivation. However, the changes in lipid levels (PC in particular) that we observe here are more drastic than the normal temporal dynamics of these lipids during blood stage progression (Gulati et al., 2015). This suggests that the perturbations are to an extent indeed the result of loss of PfGDPD. A block in PC synthesis can either cause the depletion of the PC-derived DAG pool and block TAG synthesis, or lead to increased metabolism of TAG to feed fatty acids into the compensatory Lands’ cycle that acylates lysoPC to PC (Caviglia et al., 2004; Moessinger et al., 2014). Studies in other organisms increasingly show a bidirectional link between TAG and PC synthesis in which PC-derived DAG is used for TAG synthesis and TAG-derived fatty acids are used for synthesis of PC through the Lands’ cycle (Bates and Browse, 2011; Caviglia et al., 2004; Moessinger et al., 2014; Soudant et al., 2000; van der Veen et al., 2012). The reduced haemozoin formation in GDPD-null parasites could also be a result of this decrease in TAG levels. Neutral lipids have been suggested to play a role in the parasite haem detoxification pathway and promote haemozoin formation (Hoang et al., 2010). Indeed it was recently shown that knockdown of a P. falciparum lysophospholipase results in reduced TAG levels, reduced haemozoin formation and a block in trophozoite development (Asad et al., 2021).

Our lipidomics analysis identified the betaine lipid DGTS, prevalently found in photosynthetic organisms like green algae, mosses and ferns (Giroud et al., 1988; Sato et al., 1995). DGTS has also been detected in the lipidome of Chromera velia and Vitrella brassicaformis, the algal ancestors of apicomplexan parasites (Tomčala et al., 2017) but has not been previously reported in Plasmodium. Accumulation of DGTS in choline-starved PfGDPD-null parasites is strikingly reminiscent of reports of DGTS synthesis as a non-phosphorous substitute for PC in fungi and marine bacteria under phosphate- or choline-starved conditions (Geiger et al., 2013; Riekhof et al., 2014; Sebastián et al., 2016; Senik et al., 2015). The methyl donor S-adenosyl methionine (SAM) is capable of providing both the homo-serine moiety and the methyl groups to produce DGTS from DAG (Moore et al., 2001). Enzymes involved in SAM synthesis are upregulated in lysoPC-limiting conditions and diversion of SAM pools from histone methylation towards compensatory PC biosynthetic pathways is the primary link between PC metabolism and sexual differentiation in P. falciparum (Harris et al., 2022). It is therefore tempting to speculate that blocking PC biosynthesis in P. falciparum triggers a compensatory pathway that produces DGTS as a functional substitute for PC in the parasite.

Based on protein localisation, ligand docking and sequence homology analyses, we can further speculate regarding aspects of PfGDPD function that we have not explored in this study. It has been previously suggested that the gene could use alternative start codons via ribosomal skipping to produce distinct PV-located and cytosolic variants of the protein (Denloye et al., 2012). PfGDPD could potentially perform similar functions in both compartments by facilitating the breakdown of exogenous lysoPC both within the PV and within the parasite cytosol (Brancucci et al., 2017). This scale of enzyme activity may be essential for the parasite to meet its choline needs, given the high levels of PC synthesis during parasite development and its crucial importance for intraerythrocytic membrane biogenesis. PfGDPD may also have other roles during asexual stages such as temporal and localised recycling of intracellular PC or GPC by the PfGDPD fraction expressed in the cytosol. Finally, our ligand docking simulations do not rule out additional catalytic activity towards other glycerophosphodiester substrates such as glycerophosphoethanolamine and glycerophosphoserine (Figure 6—figure supplement 2A, B). Further investigation that involves variant-specific conditional knockout of the gdpd gene could help us further dissect the role of PfGDPD in the parasite. Orthologs of PfGDPD form a Haematozoan-specific ortholog group (OG6_139464 in OrthoMCL DB release 6.4) that encompasses only blood parasites that have an intra-erythrocytic stage, that is the genera Babesia, Theileria, Plasmodium and Hepatocystis (Figure 6—figure supplement 2C). We speculate that this entire ortholog group could have a conserved role in choline acquisition for the critical process of PC biosynthesis to support an intra-erythrocytic lifestyle. PC biosynthesis is the main biosynthetic process in blood stages of Babesia (Florin-Christensen et al., 2000) and compounds that inhibit PC biosynthesis have been shown to have anti-parasite activity against Babesia and Theileria blood stages (AbouLaila et al., 2014; Gopalakrishnan et al., 2016; Maji et al., 2019; Richier et al., 2006).

In conclusion, we have demonstrated that a malaria parasite choline-releasing glycerophosphodiesterase catalyses a critical step in choline acquisition from exogenous lysoPC. Since PfGDPD-mediated procurement of choline is indispensable for normal PC biosynthesis and asexual blood stage development in the parasite, it may represent a potential new drug target.

Sequencing data have been deposited in ENA under Project PRJEB55180. All data generated or analysed are included in the manuscript or provided as source data files. All source codes are available via GitHub (https://github.com/a2g1n/GDPDxcute; copy archived at swh:1:rev:eb74fd96a9eb490579bee938d965406dbbb24769).

1. 1. Ramaprasad A

   (2022) European Nucleotide Archive

   ID PRJEB55180. A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite.

   https://www.ebi.ac.uk/ena/browser/view/PRJEB55180

1. 1. Abagyan R
   2. Totrov M

   (1994) Biased probability monte carlo conformational searches and electrostatic calculations for peptides and proteins

   Journal of Molecular Biology 235:983–1002.

   https://doi.org/10.1006/jmbi.1994.1052

   • PubMed
   • Google Scholar
2. 1. Abagyan R
   2. Totrov M
   3. Kuznetsov D

   (1994) ICM?a new method for protein modeling and design: applications to docking and structure prediction from the distorted native conformation

   Journal of Computational Chemistry 15:488–506.

   https://doi.org/10.1002/jcc.540150503

   • Google Scholar
3. 1. AbouLaila M
   2. Batadoj D
   3. Salama A
   4. Munkhjargal T
   5. Ichikawa-Seki M
   6. A Terkawi M
   7. Yokoyama N
   8. Igarashi I

   (2014) Evaluation of the inhibitory effects of miltefosine on the growth of Babesia and Theileria parasites

   Veterinary Parasitology 204:104–110.

   https://doi.org/10.1016/j.vetpar.2014.05.023

   • PubMed
   • Google Scholar
4. 1. Ancelin ML
   2. Vial HJ
   3. Philippot JR

   (1985) Inhibitors of choline transport into Plasmodium-infected erythrocytes are effective antiplasmodial compounds in vitro

   Biochemical Pharmacology 34:4068–4071.

   https://doi.org/10.1016/0006-2952(85)90390-9

   • PubMed
   • Google Scholar
5. 1. Ancelin ML
   2. Vial HJ

   (1986a) Quaternary ammonium compounds efficiently inhibit Plasmodium falciparum growth in vitro by impairment of choline transport

   Antimicrobial Agents and Chemotherapy 29:814–820.

   https://doi.org/10.1128/AAC.29.5.814

   • PubMed
   • Google Scholar
6. 1. Ancelin ML
   2. Vial HJ

   (1986b) Several lines of evidence demonstrating that Plasmodium falciparum, a parasitic organism, has distinct enzymes for the phosphorylation of choline and ethanolamine

   FEBS Letters 202:217–223.

   https://doi.org/10.1016/0014-5793(86)80690-1

   • PubMed
   • Google Scholar
7. 1. Ancelin ML
   2. Vial HJ

   (1989) Regulation of phosphatidylcholine biosynthesis in Plasmodium-infected erythrocytes

   Biochimica et Biophysica Acta 1001:82–89.

   https://doi.org/10.1016/0005-2760(89)90310-x

   • PubMed
   • Google Scholar
8. 1. Ancelin ML
   2. Calas M
   3. Vidal-Sailhan V
   4. Herbuté S
   5. Ringwald P
   6. Vial HJ

   (2003) Potent inhibitors of Plasmodium phospholipid metabolism with a broad spectrum of in vitro antimalarial activities

   Antimicrobial Agents and Chemotherapy 47:2590–2597.

   https://doi.org/10.1128/AAC.47.8.2590-2597.2003

   • PubMed
   • Google Scholar
9. 1. Asad M
   2. Yamaryo-Botté Y
   3. Hossain ME
   4. Thakur V
   5. Jain S
   6. Datta G
   7. Botté CY
   8. Mohmmed A

   (2021) An essential vesicular-trafficking phospholipase mediates neutral lipid synthesis and contributes to hemozoin formation in Plasmodium falciparum

   BMC Biology 19:159.

   https://doi.org/10.1186/s12915-021-01042-z

   • PubMed
   • Google Scholar
10. 1. Bates PD
    2. Browse J

    (2011) The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds

    The Plant Journal 68:387–399.

    https://doi.org/10.1111/j.1365-313X.2011.04693.x

    • PubMed
    • Google Scholar
11. 1. Biagini GA
    2. Pasini EM
    3. Hughes R
    4. De Koning HP
    5. Vial HJ
    6. O’Neill PM
    7. Ward SA
    8. Bray PG

    (2004) Characterization of the choline carrier of Plasmodium falciparum: a route for the selective delivery of novel antimalarial drugs

    Blood 104:3372–3377.

    https://doi.org/10.1182/blood-2004-03-1084

    • PubMed
    • Google Scholar
12. 1. Birnbaum J
    2. Flemming S
    3. Reichard N
    4. Soares AB
    5. Mesén-Ramírez P
    6. Jonscher E
    7. Bergmann B
    8. Spielmann T

    (2017) A genetic system to study Plasmodium falciparum protein function

    Nature Methods 14:450–456.

    https://doi.org/10.1038/nmeth.4223

    • PubMed
    • Google Scholar
13. 1. Botté CY
    2. Yamaryo-Botté Y
    3. Rupasinghe TWT
    4. Mullin KA
    5. MacRae JI
    6. Spurck TP
    7. Kalanon M
    8. Shears MJ
    9. Coppel RL
    10. Crellin PK
    11. Maréchal E
    12. McConville MJ
    13. McFadden GI

    (2013) Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites

    PNAS 110:7506–7511.

    https://doi.org/10.1073/pnas.1301251110

    • PubMed
    • Google Scholar
14. 1. Brancucci NMB
    2. Gerdt JP
    3. Wang C
    4. De Niz M
    5. Philip N
    6. Adapa SR
    7. Zhang M
    8. Hitz E
    9. Niederwieser I
    10. Boltryk SD
    11. Laffitte MC
    12. Clark MA
    13. Grüring C
    14. Ravel D
    15. Blancke Soares A
    16. Demas A
    17. Bopp S
    18. Rubio-Ruiz B
    19. Conejo-Garcia A
    20. Wirth DF
    21. Gendaszewska-Darmach E
    22. Duraisingh MT
    23. Adams JH
    24. Voss TS
    25. Waters AP
    26. Jiang RHY
    27. Clardy J
    28. Marti M

    (2017) Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum

    Cell 171:1532–1544.

    https://doi.org/10.1016/j.cell.2017.10.020

    • PubMed
    • Google Scholar
15. 1. Brancucci NMB
    2. De Niz M
    3. Straub TJ
    4. Ravel D
    5. Sollelis L
    6. Birren BW
    7. Voss TS
    8. Neafsey DE
    9. Marti M

    (2018) Probing Plasmodium falciparum sexual commitment at the single-cell level

    Wellcome Open Research 3:70.

    https://doi.org/10.12688/wellcomeopenres.14645.4

    • PubMed
    • Google Scholar
16. 1. Burda PC
    2. Crosskey T
    3. Lauk K
    4. Zurborg A
    5. Söhnchen C
    6. Liffner B
    7. Wilcke L
    8. Pietsch E
    9. Strauss J
    10. Jeffries CM
    11. Svergun DI
    12. Wilson DW
    13. Wilmanns M
    14. Gilberger TW

    (2020) Structure-based identification and functional characterization of a lipocalin in the malaria parasite Plasmodium falciparum

    Cell Reports 31:107817.

    https://doi.org/10.1016/j.celrep.2020.107817

    • PubMed
    • Google Scholar
17. 1. Capella-Gutiérrez S
    2. Silla-Martínez JM
    3. Gabaldón T

    (2009) TrimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses

    Bioinformatics 25:1972–1973.

    https://doi.org/10.1093/bioinformatics/btp348

    • PubMed
    • Google Scholar
18. 1. Caviglia JM
    2. De Gómez Dumm INT
    3. Coleman RA
    4. Igal RA

    (2004) Phosphatidylcholine deficiency upregulates enzymes of triacylglycerol metabolism in CHO cells

    Journal of Lipid Research 45:1500–1509.

    https://doi.org/10.1194/jlr.M400079-JLR200

    • PubMed
    • Google Scholar
19. 1. Collins CR
    2. Das S
    3. Wong EH
    4. Andenmatten N
    5. Stallmach R
    6. Hackett F
    7. Herman J-P
    8. Müller S
    9. Meissner M
    10. Blackman MJ

    (2013) Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle

    Molecular Microbiology 88:687–701.

    https://doi.org/10.1111/mmi.12206

    • PubMed
    • Google Scholar
20. 1. Contet A
    2. Pihan E
    3. Lavigne M
    4. Wengelnik K
    5. Maheshwari S
    6. Vial H
    7. Douguet D
    8. Cerdan R

    (2015) Plasmodium falciparum CTP: phosphocholine cytidylyltransferase possesses two functional catalytic domains and is inhibited by a CDP-choline analog selected from a virtual screening

    FEBS Letters 589:992–1000.

    https://doi.org/10.1016/j.febslet.2015.03.003

    • PubMed
    • Google Scholar
21. 1. Creek DJ
    2. Jankevics A
    3. Breitling R
    4. Watson DG
    5. Barrett MP
    6. Burgess KEV

    (2011) Toward global metabolomics analysis with hydrophilic interaction liquid chromatography-mass spectrometry: improved metabolite identification by retention time prediction

    Analytical Chemistry 83:8703–8710.

    https://doi.org/10.1021/ac2021823

    • PubMed
    • Google Scholar
22. 1. Davies H
    2. Belda H
    3. Broncel M
    4. Ye X
    5. Bisson C
    6. Introini V
    7. Dorin-Semblat D
    8. Semblat J-P
    9. Tibúrcio M
    10. Gamain B
    11. Kaforou M
    12. Treeck M

    (2020) An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria

    Nature Microbiology 5:848–863.

    https://doi.org/10.1038/s41564-020-0702-4

    • PubMed
    • Google Scholar
23. 1. Deerinck T
    2. Bushong E
    3. Thor A
    4. Ellisman M
    5. Thor C

    (2010)

    NCMIR methods for 3D EM: a new protocol for preparation of biological specimens for serial block face scanning electron microscopy

    Microscopy 1:6–8.

    • Google Scholar
24. 1. Denloye T
    2. Dalal S
    3. Klemba M

    (2012) Characterization of a glycerophosphodiesterase with an unusual tripartite distribution and an important role in the asexual blood stages of Plasmodium falciparum

    Molecular and Biochemical Parasitology 186:29–37.

    https://doi.org/10.1016/j.molbiopara.2012.09.004

    • PubMed
    • Google Scholar
25. 1. Dushianthan A
    2. Cusack R
    3. Koster G
    4. Grocott MPW
    5. Postle AD

    (2019) Insight into erythrocyte phospholipid molecular flux in healthy humans and in patients with acute respiratory distress syndrome

    PLOS ONE 14:e0221595.

    https://doi.org/10.1371/journal.pone.0221595

    • PubMed
    • Google Scholar
26. 1. Edgar RC

    (2004) Muscle: a multiple sequence alignment method with reduced time and space complexity

    BMC Bioinformatics 5:113.

    https://doi.org/10.1186/1471-2105-5-113

    • PubMed
    • Google Scholar
27. 1. Fernández-Murray JP
    2. McMaster CR

    (2005) Glycerophosphocholine catabolism as a new route for choline formation for phosphatidylcholine synthesis by the Kennedy pathway

    The Journal of Biological Chemistry 280:38290–38296.

    https://doi.org/10.1074/jbc.M507700200

    • PubMed
    • Google Scholar
28. 1. Florin-Christensen J
    2. Suarez CE
    3. Florin-Christensen M
    4. Hines SA
    5. McElwain TF
    6. Palmer GH

    (2000) Phosphatidylcholine formation is the predominant lipid biosynthetic event in the hemoparasite Babesia bovis

    Molecular and Biochemical Parasitology 106:147–156.

    https://doi.org/10.1016/s0166-6851(99)00209-1

    • PubMed
    • Google Scholar
29. 1. Garcia-Gonzalo FR
    2. Izpisúa Belmonte JC

    (2008) Albumin-associated lipids regulate human embryonic stem cell self-renewal

    PLOS ONE 3:e1384.

    https://doi.org/10.1371/journal.pone.0001384

    • PubMed
    • Google Scholar
30. 1. Geiger O
    2. López-Lara IM
    3. Sohlenkamp C

    (2013) Phosphatidylcholine biosynthesis and function in bacteria

    Biochimica et Biophysica Acta 1831:503–513.

    https://doi.org/10.1016/j.bbalip.2012.08.009

    • PubMed
    • Google Scholar
31. 1. Ghorbal M
    2. Gorman M
    3. Macpherson CR
    4. Martins RM
    5. Scherf A
    6. Lopez-Rubio JJ

    (2014) Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system

    Nature Biotechnology 32:819–821.

    https://doi.org/10.1038/nbt.2925

    • PubMed
    • Google Scholar
32. 1. Giroud C
    2. Gerber A
    3. Eichenberger W

    (1988) Lipids of Chlamydomonas reinhardtii: analysis of molecular species and intracellular site(s) of biosynthesis

    Plant and Cell Physiology 29:587–595.

    https://doi.org/10.1093/oxfordjournals.pcp.a077533

    • Google Scholar
33. 1. González-Bulnes P
    2. Bobenchik AM
    3. Augagneur Y
    4. Cerdan R
    5. Vial HJ
    6. Llebaria A
    7. Ben Mamoun C

    (2011) PG12, a phospholipid analog with potent antimalarial activity, inhibits Plasmodium falciparum CTP: phosphocholine cytidylyltransferase activity

    The Journal of Biological Chemistry 286:28940–28947.

    https://doi.org/10.1074/jbc.M111.268946

    • PubMed
    • Google Scholar
34. 1. Gopalakrishnan A
    2. Maji C
    3. Dahiya RK
    4. Suthar A
    5. Kumar R
    6. Gupta AK
    7. Dimri U
    8. Kumar S

    (2016) In vitro growth inhibitory efficacy of some target specific novel drug molecules against Theileria equi

    Veterinary Parasitology 217:1–6.

    https://doi.org/10.1016/j.vetpar.2015.12.024

    • PubMed
    • Google Scholar
35. 1. Greenwood DJ
    2. Dos Santos MS
    3. Huang S
    4. Russell MRG
    5. Collinson LM
    6. MacRae JI
    7. West A
    8. Jiang H
    9. Gutierrez MG

    (2019) Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected macrophages

    Science 364:1279–1282.

    https://doi.org/10.1126/science.aat9689

    • PubMed
    • Google Scholar
36. 1. Gulati S
    2. Ekland EH
    3. Ruggles KV
    4. Chan RB
    5. Jayabalasingham B
    6. Zhou B
    7. Mantel P-Y
    8. Lee MCS
    9. Spottiswoode N
    10. Coburn-Flynn O
    11. Hjelmqvist D
    12. Worgall TS
    13. Marti M
    14. Di Paolo G
    15. Fidock DA

    (2015) Profiling the essential nature of lipid metabolism in asexual blood and gametocyte stages of Plasmodium falciparum

    Cell Host & Microbe 18:371–381.

    https://doi.org/10.1016/j.chom.2015.08.003

    • PubMed
    • Google Scholar
37. 1. Harris PK
    2. Yeoh S
    3. Dluzewski AR
    4. O’Donnell RA
    5. Withers-Martinez C
    6. Hackett F
    7. Bannister LH
    8. Mitchell GH
    9. Blackman MJ

    (2005) Molecular identification of a malaria merozoite surface sheddase

    PLOS Pathogens 1:e0010029.

    https://doi.org/10.1371/journal.ppat.0010029

    • PubMed
    • Google Scholar
38. Preprint

    1. Harris CT
    2. Tong X
    3. Campelo R
    4. Marreiros IM
    5. Vanheer LN
    6. Nahiyaan N
    7. Zuzarte-Luís VA
    8. Deitsch KW
    9. Mota MM
    10. Rhee KY
    11. Kafsack BFC

    (2022) Metabolic Competition between Lipid Metabolism and Histone Methylation Regulates Sexual Differentiation in Human Malaria Parasites

    bioRxiv.

    https://doi.org/10.1101/2022.01.18.476397

    • Google Scholar
39. 1. Hoang AN
    2. Sandlin RD
    3. Omar A
    4. Egan TJ
    5. Wright DW

    (2010) The neutral lipid composition present in the digestive vacuole of Plasmodium falciparum concentrates heme and mediates β-hematin formation with an unusually low activation energy

    Biochemistry 49:10107–10116.

    https://doi.org/10.1021/bi101397u

    • PubMed
    • Google Scholar
40. 1. Hulo N
    2. Bairoch A
    3. Bulliard V
    4. Cerutti L
    5. Cuche BA
    6. de Castro E
    7. Lachaize C
    8. Langendijk-Genevaux PS
    9. Sigrist CJA

    (2008) The 20 years of prosite

    Nucleic Acids Research 36:D245–D249.

    https://doi.org/10.1093/nar/gkm977

    • PubMed
    • Google Scholar
41. 1. Jones P
    2. Binns D
    3. Chang HY
    4. Fraser M
    5. Li W
    6. McAnulla C
    7. McWilliam H
    8. Maslen J
    9. Mitchell A
    10. Nuka G
    11. Pesseat S
    12. Quinn AF
    13. Sangrador-Vegas A
    14. Scheremetjew M
    15. Yong SY
    16. Lopez R
    17. Hunter S

    (2014) InterProScan 5: genome-scale protein function classification

    Bioinformatics 30:1236–1240.

    https://doi.org/10.1093/bioinformatics/btu031

    • PubMed
    • Google Scholar
42. 1. Jones ML
    2. Das S
    3. Belda H
    4. Collins CR
    5. Blackman MJ
    6. Treeck M

    (2016) A versatile strategy for rapid conditional genome engineering using loxp sites in A small synthetic intron in Plasmodium falciparum

    Scientific Reports 6:21800.

    https://doi.org/10.1038/srep21800

    • PubMed
    • Google Scholar
43. 1. Jumper J
    2. Evans R
    3. Pritzel A
    4. Green T
    5. Figurnov M
    6. Ronneberger O
    7. Tunyasuvunakool K
    8. Bates R
    9. Žídek A
    10. Potapenko A
    11. Bridgland A
    12. Meyer C
    13. Kohl SAA
    14. Ballard AJ
    15. Cowie A
    16. Romera-Paredes B
    17. Nikolov S
    18. Jain R
    19. Adler J
    20. Back T
    21. Petersen S
    22. Reiman D
    23. Clancy E
    24. Zielinski M
    25. Steinegger M
    26. Pacholska M
    27. Berghammer T
    28. Bodenstein S
    29. Silver D
    30. Vinyals O
    31. Senior AW
    32. Kavukcuoglu K
    33. Kohli P
    34. Hassabis D

    (2021) Highly accurate protein structure prediction with alphafold

    Nature 596:583–589.

    https://doi.org/10.1038/s41586-021-03819-2

    • PubMed
    • Google Scholar
44. 1. Kilian N
    2. Choi JY
    3. Voelker DR
    4. Ben Mamoun C

    (2018) Role of phospholipid synthesis in the development and differentiation of malaria parasites in the blood

    The Journal of Biological Chemistry 293:17308–17316.

    https://doi.org/10.1074/jbc.R118.003213

    • PubMed
    • Google Scholar
45. 1. Kirk K
    2. Wong HY
    3. Elford BC
    4. Newbold CI
    5. Ellory JC

    (1991) Enhanced choline and rb+ transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum

    The Biochemical Journal 278 ( Pt 2):521–525.

    https://doi.org/10.1042/bj2780521

    • PubMed
    • Google Scholar
46. 1. Knuepfer E
    2. Napiorkowska M
    3. van Ooij C
    4. Holder AA

    (2017) Generating conditional gene knockouts in plasmodium - a toolkit to produce stable dicre recombinase-expressing parasite lines using CRISPR/cas9

    Scientific Reports 7:3881.

    https://doi.org/10.1038/s41598-017-03984-3

    • PubMed
    • Google Scholar
47. 1. Koelmel JP
    2. Kroeger NM
    3. Ulmer CZ
    4. Bowden JA
    5. Patterson RE
    6. Cochran JA
    7. Beecher CWW
    8. Garrett TJ
    9. Yost RA

    (2017) LipidMatch: an automated workflow for rule-based lipid identification using untargeted high-resolution tandem mass spectrometry data

    BMC Bioinformatics 18:331.

    https://doi.org/10.1186/s12859-017-1744-3

    • PubMed
    • Google Scholar
48. 1. Letunic I
    2. Bork P

    (2021) Interactive tree of life (itol) V5: an online tool for phylogenetic tree display and annotation

    Nucleic Acids Research 49:W293–W296.

    https://doi.org/10.1093/nar/gkab301

    • PubMed
    • Google Scholar
49. 1. Maji C
    2. Goel P
    3. Suthar A
    4. Mandal KD
    5. Gopalakrishnan A
    6. Kumar R
    7. Tripathi BN
    8. Kumar S

    (2019) Lumefantrine and o-choline-parasite metabolism specific drug molecules inhibited in vitro growth of Theileria equi and Babesia caballi in MASP culture system

    Ticks and Tick-Borne Diseases 10:568–574.

    https://doi.org/10.1016/j.ttbdis.2019.01.004

    • PubMed
    • Google Scholar
50. 1. Malleret B
    2. Claser C
    3. Ong ASM
    4. Suwanarusk R
    5. Sriprawat K
    6. Howland SW
    7. Russell B
    8. Nosten F
    9. Rénia L

    (2011) A rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development

    Scientific Reports 1:118.

    https://doi.org/10.1038/srep00118

    • PubMed
    • Google Scholar
51. 1. Mesén-Ramírez P
    2. Reinsch F
    3. Blancke Soares A
    4. Bergmann B
    5. Ullrich AK
    6. Tenzer S
    7. Spielmann T

    (2016) Stable translocation intermediates jam global protein export in Plasmodium falciparum parasites and link the PTEX component exp2 with translocation activity

    PLOS Pathogens 12:e1005618.

    https://doi.org/10.1371/journal.ppat.1005618

    • PubMed
    • Google Scholar
52. 1. Mesén-Ramírez P
    2. Bergmann B
    3. Tran TT
    4. Garten M
    5. Stäcker J
    6. Naranjo-Prado I
    7. Höhn K
    8. Zimmerberg J
    9. Spielmann T

    (2019) EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites

    PLOS Biology 17:e3000473.

    https://doi.org/10.1371/journal.pbio.3000473

    • PubMed
    • Google Scholar
53. 1. Moessinger C
    2. Klizaite K
    3. Steinhagen A
    4. Philippou-Massier J
    5. Shevchenko A
    6. Hoch M
    7. Ejsing CS
    8. Thiele C

    (2014) Two different pathways of phosphatidylcholine synthesis, the kennedy pathway and the lands cycle, differentially regulate cellular triacylglycerol storage

    BMC Cell Biology 15:43.

    https://doi.org/10.1186/s12860-014-0043-3

    • PubMed
    • Google Scholar
54. 1. Moon RW
    2. Hall J
    3. Rangkuti F
    4. Ho YS
    5. Almond N
    6. Mitchell GH
    7. Pain A
    8. Holder AA
    9. Blackman MJ

    (2013) Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

    PNAS 110:531–536.

    https://doi.org/10.1073/pnas.1216457110

    • PubMed
    • Google Scholar
55. 1. Moore TS
    2. Du Z
    3. Chen Z

    (2001) Membrane lipid biosynthesis in Chlamydomonas reinhardtii: in vitro biosynthesis of diacylglyceryltrimethylhomoserine

    Plant Physiology 125:423–429.

    https://doi.org/10.1104/pp.125.1.423

    • PubMed
    • Google Scholar
56. 1. Morita J
    2. Kano K
    3. Kato K
    4. Takita H
    5. Sakagami H
    6. Yamamoto Y
    7. Mihara E
    8. Ueda H
    9. Sato T
    10. Tokuyama H
    11. Arai H
    12. Asou H
    13. Takagi J
    14. Ishitani R
    15. Nishimasu H
    16. Nureki O
    17. Aoki J

    (2016) Structure and biological function of ENPP6, a choline-specific glycerophosphodiester-phosphodiesterase

    Scientific Reports 6:20995.

    https://doi.org/10.1038/srep20995

    • PubMed
    • Google Scholar
57. 1. Palacpac NMQ
    2. Hiramine Y
    3. Mi-ichi F
    4. Torii M
    5. Kita K
    6. Hiramatsu R
    7. Horii T
    8. Mitamura T

    (2004) Developmental-Stage-Specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies in Plasmodium falciparum-infected erythrocytes

    Journal of Cell Science 117:1469–1480.

    https://doi.org/10.1242/jcs.00988

    • PubMed
    • Google Scholar
58. 1. Perrin AJ
    2. Collins CR
    3. Russell MRG
    4. Collinson LM
    5. Baker DA
    6. Blackman MJ

    (2018) The actinomyosin motor drives malaria parasite red blood cell invasion but not egress

    MBio 9:e00905-18.

    https://doi.org/10.1128/mBio.00905-18

    • PubMed
    • Google Scholar
59. 1. Pessi G
    2. Kociubinski G
    3. Mamoun CB

    (2004) A pathway for phosphatidylcholine biosynthesis in Plasmodium falciparum involving phosphoethanolamine methylation

    PNAS 101:6206–6211.

    https://doi.org/10.1073/pnas.0307742101

    • PubMed
    • Google Scholar
60. Software

    1. Ramaprasad A

    (2022) GDPDxcute, version swh:1:rev:eb74fd96a9eb490579bee938d965406dbbb24769

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:dir:30bdd60e2ccb176c9b137d958b4838e77da19504;origin=https://github.com/a2g1n/GDPDxcute;visit=swh:1:snp:96e08fbfc52b5e8a568ab53474a4ad2c8b2a4741;anchor=swh:1:rev:eb74fd96a9eb490579bee938d965406dbbb24769
61. 1. Rao KN
    2. Bonanno JB
    3. Burley SK
    4. Swaminathan S

    (2006) Crystal structure of glycerophosphodiester phosphodiesterase from Agrobacterium tumefaciens by sad with a large asymmetric unit

    Proteins 65:514–518.

    https://doi.org/10.1002/prot.21079

    • PubMed
    • Google Scholar
62. Software

    1. R Development Core Team

    (2021) R: A language and environment for statistical computing

    R Foundation for Statistical Computing, Vienna, Austria.

    https://www.r-project.org/index.html
63. 1. Richier E
    2. Biagini GA
    3. Wein S
    4. Boudou F
    5. Bray PG
    6. Ward SA
    7. Precigout E
    8. Calas M
    9. Dubremetz JF
    10. Vial HJ

    (2006) Potent antihematozoan activity of novel bisthiazolium drug T16: evidence for inhibition of phosphatidylcholine metabolism in erythrocytes infected with Babesia and Plasmodium spp

    Antimicrobial Agents and Chemotherapy 50:3381–3388.

    https://doi.org/10.1128/AAC.00443-06

    • PubMed
    • Google Scholar
64. 1. Riekhof WR
    2. Naik S
    3. Bertrand H
    4. Benning C
    5. Voelker DR

    (2014) Phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus-free betaine lipid diacylglyceryl-N, N, N-trimethylhomoserine

    Eukaryotic Cell 13:749–757.

    https://doi.org/10.1128/EC.00004-14

    • PubMed
    • Google Scholar
65. 1. Robinson JT
    2. Thorvaldsdóttir H
    3. Winckler W
    4. Guttman M
    5. Lander ES
    6. Getz G
    7. Mesirov JP

    (2011) Integrative genomics viewer

    Nature Biotechnology 29:24–26.

    https://doi.org/10.1038/nbt.1754

    • PubMed
    • Google Scholar
66. 1. Sato N
    2. Tsuzuki M
    3. Matsuda Y
    4. Ehara T
    5. Osafune T
    6. Kawaguchi A

    (1995) Isolation and characterization of mutants affected in lipid metabolism of Chlamydomonas reinhardtii

    European Journal of Biochemistry 230:987–993.

    https://doi.org/10.1111/j.1432-1033.1995.tb20646.x

    • PubMed
    • Google Scholar
67. 1. Schneider CA
    2. Rasband WS
    3. Eliceiri KW

    (2012) Nih image to imagej: 25 years of image analysis

    Nature Methods 9:671–675.

    https://doi.org/10.1038/nmeth.2089

    • PubMed
    • Google Scholar
68. 1. Sebastián M
    2. Smith AF
    3. González JM
    4. Fredricks HF
    5. Van Mooy B
    6. Koblížek M
    7. Brandsma J
    8. Koster G
    9. Mestre M
    10. Mostajir B
    11. Pitta P
    12. Postle AD
    13. Sánchez P
    14. Gasol JM
    15. Scanlan DJ
    16. Chen Y

    (2016) Lipid remodelling is a widespread strategy in marine heterotrophic bacteria upon phosphorus deficiency

    The ISME Journal 10:968–978.

    https://doi.org/10.1038/ismej.2015.172

    • PubMed
    • Google Scholar
69. 1. Senik SV
    2. Maloshenok LG
    3. Kotlova ER
    4. Shavarda AL
    5. Moiseenko KV
    6. Bruskin SA
    7. Koroleva OV
    8. Psurtseva NV

    (2015) Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes

    Phytochemistry 117:34–42.

    https://doi.org/10.1016/j.phytochem.2015.05.021

    • PubMed
    • Google Scholar
70. 1. Serrán-Aguilera L
    2. Denton H
    3. Rubio-Ruiz B
    4. López-Gutiérrez B
    5. Entrena A
    6. Izquierdo L
    7. Smith TK
    8. Conejo-García A
    9. Hurtado-Guerrero R

    (2016) Plasmodium falciparum choline kinase inhibition leads to a major decrease in phosphatidylethanolamine causing parasite death

    Scientific Reports 6:33189.

    https://doi.org/10.1038/srep33189

    • PubMed
    • Google Scholar
71. 1. Shi L
    2. Liu JF
    3. An XM
    4. Liang DC

    (2008) Crystal structure of glycerophosphodiester phosphodiesterase (GDPD) from Thermoanaerobacter tengcongensis, a metal ion-dependent enzyme: insight into the catalytic mechanism

    Proteins 72:280–288.

    https://doi.org/10.1002/prot.21921

    • PubMed
    • Google Scholar
72. 1. Soudant P
    2. Chu FL
    3. Marty Y

    (2000) Lipid class composition of the protozoan Perkinsus marinus, an oyster parasite, and its metabolism of a fluorescent phosphatidylcholine analog

    Lipids 35:1387–1395.

    https://doi.org/10.1007/s11745-000-0656-1

    • PubMed
    • Google Scholar
73. 1. Stamatakis A

    (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies

    Bioinformatics 30:1312–1313.

    https://doi.org/10.1093/bioinformatics/btu033

    • PubMed
    • Google Scholar
74. 1. Stewart JD
    2. Marchan R
    3. Lesjak MS
    4. Lambert J
    5. Hergenroeder R
    6. Ellis JK
    7. Lau CH
    8. Keun HC
    9. Schmitz G
    10. Schiller J
    11. Eibisch M
    12. Hedberg C
    13. Waldmann H
    14. Lausch E
    15. Tanner B
    16. Sehouli J
    17. Sagemueller J
    18. Staude H
    19. Steiner E
    20. Hengstler JG

    (2012) Choline-releasing glycerophosphodiesterase EDI3 drives tumor cell migration and metastasis

    PNAS 109:8155–8160.

    https://doi.org/10.1073/pnas.1117654109

    • PubMed
    • Google Scholar
75. 1. Thomas JA
    2. Collins CR
    3. Das S
    4. Hackett F
    5. Graindorge A
    6. Bell D
    7. Deu E
    8. Blackman MJ

    (2016) Development and application of a simple plaque assay for the human malaria parasite Plasmodium falciparum

    PLOS ONE 11:e0157873.

    https://doi.org/10.1371/journal.pone.0157873

    • PubMed
    • Google Scholar
76. 1. Tibúrcio M
    2. Yang ASP
    3. Yahata K
    4. Suárez-Cortés P
    5. Belda H
    6. Baumgarten S
    7. van de Vegte-Bolmer M
    8. van Gemert GJ
    9. van Waardenburg Y
    10. Levashina EA
    11. Sauerwein RW
    12. Treeck M

    (2019) A novel tool for the generation of conditional knockouts to study gene function across the Plasmodium falciparum life cycle

    MBio 10:e01170-19.

    https://doi.org/10.1128/mBio.01170-19

    • PubMed
    • Google Scholar
77. 1. Tomčala A
    2. Kyselová V
    3. Schneedorferová I
    4. Opekarová I
    5. Moos M
    6. Urajová P
    7. Kručinská J
    8. Oborník M

    (2017) Separation and identification of lipids in the photosynthetic cousins of Apicomplexa Chromera velia and vitrella brassicaformis

    Journal of Separation Science 40:3402–3413.

    https://doi.org/10.1002/jssc.201700171

    • PubMed
    • Google Scholar
78. 1. Trager W
    2. Jensen JB

    (1976) Human malaria parasites in continuous culture

    Science 193:673–675.

    https://doi.org/10.1126/science.781840

    • PubMed
    • Google Scholar
79. 1. Tsugawa H
    2. Cajka T
    3. Kind T
    4. Ma Y
    5. Higgins B
    6. Ikeda K
    7. Kanazawa M
    8. VanderGheynst J
    9. Fiehn O
    10. Arita M

    (2015) MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis

    Nature Methods 12:523–526.

    https://doi.org/10.1038/nmeth.3393

    • PubMed
    • Google Scholar
80. 1. van der Veen JN
    2. Lingrell S
    3. Vance DE

    (2012) The membrane lipid phosphatidylcholine is an unexpected source of triacylglycerol in the liver

    The Journal of Biological Chemistry 287:23418–23426.

    https://doi.org/10.1074/jbc.M112.381723

    • PubMed
    • Google Scholar
81. 1. van Ooij C
    2. Withers-Martinez C
    3. Ringel A
    4. Cockcroft S
    5. Haldar K
    6. Blackman MJ

    (2013) Identification of a Plasmodium falciparum phospholipid transfer protein

    The Journal of Biological Chemistry 288:31971–31983.

    https://doi.org/10.1074/jbc.M113.474189

    • PubMed
    • Google Scholar
82. 1. Varadi M
    2. Anyango S
    3. Deshpande M
    4. Nair S
    5. Natassia C
    6. Yordanova G
    7. Yuan D
    8. Stroe O
    9. Wood G
    10. Laydon A
    11. Žídek A
    12. Green T
    13. Tunyasuvunakool K
    14. Petersen S
    15. Jumper J
    16. Clancy E
    17. Green R
    18. Vora A
    19. Lutfi M
    20. Figurnov M
    21. Cowie A
    22. Hobbs N
    23. Kohli P
    24. Kleywegt G
    25. Birney E
    26. Hassabis D
    27. Velankar S

    (2022) AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models

    Nucleic Acids Research 50:D439–D444.

    https://doi.org/10.1093/nar/gkab1061

    • PubMed
    • Google Scholar
83. 1. Vial HJ
    2. Thuet MJ
    3. Philippot JR

    (1984) Cholinephosphotransferase and ethanolaminephosphotransferase activities in Plasmodium knowlesi-infected erythrocytes: their use as parasite-specific markers

    Biochimica et Biophysica Acta 795:372–383.

    https://doi.org/10.1016/0005-2760(84)90088-2

    • PubMed
    • Google Scholar
84. 1. Vielemeyer O
    2. McIntosh MT
    3. Joiner KA
    4. Coppens I

    (2004) Neutral lipid synthesis and storage in the intraerythrocytic stages of Plasmodium falciparum

    Molecular and Biochemical Parasitology 135:197–209.

    https://doi.org/10.1016/j.molbiopara.2003.08.017

    • PubMed
    • Google Scholar
85. 1. Wein S
    2. Ghezal S
    3. Buré C
    4. Maynadier M
    5. Périgaud C
    6. Vial HJ
    7. Lefebvre-Tournier I
    8. Wengelnik K
    9. Cerdan R

    (2018) Contribution of the precursors and interplay of the pathways in the phospholipid metabolism of the malaria parasite

    Journal of Lipid Research 59:1461–1471.

    https://doi.org/10.1194/jlr.M085589

    • PubMed
    • Google Scholar
86. 1. Witola WH
    2. El Bissati K
    3. Pessi G
    4. Xie C
    5. Roepe PD
    6. Mamoun CB

    (2008) Disruption of the Plasmodium falciparum PfPMT gene results in a complete loss of phosphatidylcholine biosynthesis via the serine-decarboxylase-phosphoethanolamine-methyltransferase pathway and severe growth and survival defects

    The Journal of Biological Chemistry 283:27636–27643.

    https://doi.org/10.1074/jbc.M804360200

    • PubMed
    • Google Scholar
87. 1. Zhang M
    2. Wang C
    3. Otto TD
    4. Oberstaller J
    5. Liao X
    6. Adapa SR
    7. Udenze K
    8. Bronner IF
    9. Casandra D
    10. Mayho M
    11. Brown J
    12. Li S
    13. Swanson J
    14. Rayner JC
    15. Jiang RHY
    16. Adams JH

    (2018) Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis

    Science 360:eaap7847.

    https://doi.org/10.1126/science.aap7847

    • PubMed
    • Google Scholar

Decision letter after peer review:

Thank you for submitting your article "A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Dominique Soldati-Favre as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Paul A Sigala (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Provide additional panel in Figure 2 confirming localization of PfGDPH with a food vacuole marker

2) Provide further evidence for how exogenous choline rescues GDPD KO parasite lipid levels, ideally via isotope tracing to unambiguously demonstrate utilization of exogenous choline rather than upregulation/utilization of an alternative source.

3) Revise Figure 5 (especially 5C) to clarify the experimental set-up and better match to textual descriptions

4) Discuss or confirm alternative possibilities for role of GDPH in gametocytogenesis

Reviewer #1 (Recommendations for the authors):

The gene knock out studies, as well as enzyme activity analysis of PfGDPD were carried out in an earlier study (Denloye et al., 2012), which highlighted its role in parasite survival and possible role in providing choline for phospholipid synthesis in the parasite.

The phospholipid analysis shows a generalized response of disturbance in lipid homeostasis; phospholipid analysis in the first cycle (+RAP/-RAP) shows effect on levels of several phospholipids which are overall down, and a generalized increase in DAGs and TAGs. There is a clear mis-regulation of lipid metabolism in the GDPD-KO conditions, but it seems unclear how exactly it is linked with its proposed functions. The authors highlighted that the parasite can shift from CDP-choline pathway to the SDPM pathway to produce PC, which could be reason for lowered PE and PS production; however, the PS production is independent of PC or PE synthesis, which may suggest generalized effect on phospholipid profiles. Have the authors assessed upregulation of PMT pathway under KO conditions? What is the explanation for DAG and TAG increase under KO conditions?

In the cell biology study of the KO line, parasites show developmental arrest at trophozoite, reduction in merozoite number as well as effect on food vacuole functioning resulting in decreased haemozoin crystal. These could be generalised stress response and no direct correlation is established with the GDPD reduction. Why haemozoin crystal formation is hindered in the KO parasites, and how is it linked with disturbance in lipid metabolism and specifically PC synthesis?

Rescue of KO parasites with choline is intriguing but somewhat inexplicable. In the parasite, majority of PC is synthesised using choline derived from lysoPC, whereas under LPC depleted condition the parasite is shown to utilize PMT pathway for PC synthesis by compensating PE synthesis, rather than utilizing available choline in the serum. Even if some PC gets synthesized using choline taken from milieu, it should be able to recover parasite growth to only a certain level, however, in this study major recovery of parasite growth is shown. Mechanistic proof of choline rescue is missing. It is important to assess phospholipid profile to show normalized PC (and other phospholipids) synthesis under choline rescue condition. The authors should use labelled choline to show its incorporation in the PC synthesised under rescue condition. Can excess glycerophosphocholine (GPC) rescue the parasites under partial knock-down conditions?

Other comments:

– I did not get the supplementary figures on the website or in the BioRxiv site.

– Figure 4B: the parasitaemia is marked in log scale which is confusing to compare between different sets.

– Figure 5: Why LPCs are getting accumulated in the KO, which are targets of Lysophospholipase, whereas the direct target of GDPD is Glycero-phosphocholine. What is the status of glycero-phosphocholine in these parasites?

– Figure 5C: The labelled proportion of the majority of PC species is not significantly different between the two clones.

– The experimental setup for Figure 5C is confusing, the G1 parasite strain is expected to be completely devoid of PfGDPD, if the GDPD is only enzyme responsible for release of choline from GPC, then how can this parasite utilize lyoPC for labelling the newly synthesised PC.

– What is the significance of betaine lipid DGTS upregulation, DGTS is synthesised without the use of choline but using DAGs, how does this data correlate with other lipidomic profile, DAG levels as well as with the choline rescue.

Reviewer #2 (Recommendations for the authors):

1. Is GDPD localization to the PV and/or cytoplasm critical for parasite viability? Some discussion of this question seems warranted at a minimum. This key question could be strongly addressed if the authors extend the experiment in Figure 2G to test if episomal expression of GDPD variants either lacking the SP (and thus presumably just cytoplasmic) or lacking Met19 (which may prevent a cytoplasmic population and just retain the PV population) rescue parasites from loss of endogenous GDPD.

2. Do the GDPD mutations studied episomally in Figure 2G impact or reduce protein stability and expression? The microscopy data in the figure supplement suggest that all proteins are expressed but quantitative comparison is difficult. Can the authors show by western blot (normalized to loading control) that WT and enzyme mutants are all expressed at similar levels? This experiment seems critical since the 2012 Klemba study reported that a different active site mutant resulted in insoluble protein.

3. Is the mechanism of choline uptake into parasites known? Are New Permeability Pathway mechanisms involved (e.g., does furosemide antagonize uptake?)?

4. For the experiment in Figure 5A, does addition of exogenous choline to +RAP parasites lead to phospholipid levels that phenocopy -RAP conditions? This experiment may give insight into other GDPD functions beyond choline-dependent phospholipid synthesis.

5. The current labeling in Figure 5C is confusing and difficult to match to the relevant textual descriptions, as this same panel is referenced in lines 250 and 259 in different Results sections. The Figure 5C graph on the right (specific PC labeling) is also described in the text before the left graph, which adds confusion. It would be helpful to explicitly label the left graph as total lipids and possibly label the left/right graphs as distinct panels C and D that can be referred to separately and matched with the relevant text descriptions.

6. The 2012 Klemba paper previously showed that recombinant GDPD could hydrolyze glycerophosphocholine. Figure 6 in the present paper is a valuable extension of this experiment to endogenous GDPD from parasites, and these experiments are important to strongly support the proposed enzyme function in choline mobilization. Comparison of IP samples of GDPD {plus minus}RAP or to stable GDPD-deletion clone G1 provides some but incomplete support for specific function, as there could also be other functional losses in parasites that accompany GDPD deletion or co-purifying proteins that accompany GDPD pull-down that impact choline release in these experiments. Do anti-mCherry IPs under +RAP conditions of the parasites complemented with WT but not mutant enzymes (in Figure 2G) phenocopy GPC and choline levels in B4- parasites, as expected if differences between B4- and B4+ or G1 parasites are due to the specific activity of GDPD?

Reviewer #3 (Recommendations for the authors):

– The catalytic function of this protein has already been investigated in Denloye et al., 2012, this figure should be removed and instead the data from this previous article can be discussed.

– Not all defects, especially at the lipid level, can be explained with the current data and their interpretations: Why Do TAGs and DAGs increase in the mutant? Is there a link between accumulation of hemozoin crystals and neutral lipid accumulation, as recently reported (Asad et al. BMC Biology), have the author tested it? How do the authors explain that choline complementation rescues the parasite when this chline is believed to exclusively being generated from LPC?

– Some graphs don't have any error bars.

– Statistical significance is not always shown, and the tests performed are not discussed in the figure captions.

– Representation of the lipidomic data is often hard to follow, individual FA species of each lipid group is only shown in the supplementary figures, which also do not show the statistical significance of the data; were any stat tests done?

– No statistical significance is shown in Figure 5B and C.

– I would propose to the authors to replace the bubble charts that is quite difficult to read and interpret, with only the significant results of PE, PS, PC, LPC and DAG in the form of the bar graphs shown in Figure 5—figure supplement 1.

– In the PfGDPD-null mutant it would of interest to see if the expression of other transcripts has changed, and this can expand on how this parasite can survive solely on the exogenous choline despite having completely lost this proposed essential protein.

– Transcriptional analyses will also reveal why despite the LPC/PC balance being offset, sexual conversion is not induced.

– The lipidomic analyses is extremely detailed and it seems to be overly condensed to a single image and not fully deliberated in the discussion.

– What is the relation between the accumulation of DGTS and DAG? This could be addressed in the discussion of the paper.

Essential revisions:

1) Provide additional panel in Figure 2 confirming localization of PfGDPH with a food vacuole marker

2) Provide further evidence for how exogenous choline rescues GDPD KO parasite lipid levels, ideally via isotope tracing to unambiguously demonstrate utilization of exogenous choline rather than upregulation/utilization of an alternative source.

3) Revise Figure 5 (especially 5C) to clarify the experimental set-up and better match to textual descriptions

4) Discuss or confirm alternative possibilities for role of GDPH in gametocytogenesis

We thank the editor for this summary of the revisions considered essential for this manuscript. In the revised manuscript we have now dealt specifically with these 4 requests, as laid out in detail below. Note that we assume that for Essential Revision #1 above, the editor means the ‘parasitophorous vacuole’ rather than the ‘food vacuole’, which was not requested by the Reviewers.

Reviewer #1 (Recommendations for the authors):

The gene knock out studies, as well as enzyme activity analysis of PfGDPD were carried out in an earlier study (Denloye et al., 2012), which highlighted its role in parasite survival and possible role in providing choline for phospholipid synthesis in the parasite.

The phospholipid analysis shows a generalized response of disturbance in lipid homeostasis; phospholipid analysis in the first cycle (+RAP/-RAP) shows effect on levels of several phospholipids which are overall down, and a generalized increase in DAGs and TAGs. There is a clear mis-regulation of lipid metabolism in the GDPD-KO conditions, but it seems unclear how exactly it is linked with its proposed functions. The authors highlighted that the parasite can shift from CDP-choline pathway to the SDPM pathway to produce PC, which could be reason for lowered PE and PS production; however, the PS production is independent of PC or PE synthesis, which may suggest generalized effect on phospholipid profiles. Have the authors assessed upregulation of PMT pathway under KO conditions? What is the explanation for DAG and TAG increase under KO conditions?

We completely agree with the implied view of the Reviewer that our first lipidomics analysis, that of RAP+ and RAP- parasites at schizont stage in cycle 0 (original Figure 5A), whilst revealing a reduction in key structural phospholipids, does not reliably inform on the function of GDPD. While we chose this time point as a starting point for the lipidomics analysis for the reasons detailed (see lines 203-211 of the original submission), we initially underestimated the confounding effects of residual GDPD activity still present in the RAP+ parasites at the end of cycle 0 (original Figure 2D). Even those residual levels of GDPD can break down GPC to choline (Figure 6), likely explaining why our first lipidomics experiment revealed only a small and non-significant decrease in lysoPC-derived choline incorporation into PC (original Figure 5B). This, we suspect, combined with the compensatory SDPM pathway, resulted in unchanged PC levels in the cycle 0 schizonts. The ambiguity inherent in this experiment therefore motivated us to repeat the lipidomic analysis with the completely GDPD-null clonal line G1, following prolonged maintenance in culture (original Figure 5C), ultimately leading to a much clearer picture of GDPD function.

Although we did not directly assess upregulation of PMT activity in the GDPD-null G1 parasites, we believe that the reduction in PE and PS levels detected reflects activation of the SDPM pathway. This is because we detected a significant reduction in only PS, PE and π levels and not an overall decrease in all PLs (PC and PG are essentially unaffected). The reviewer is correct in stating that PS synthesis is independent of PC or PE synthesis under normal conditions, but upon activation of the SDPM pathway both serine and ethanolamine are expected to be redirected towards PC synthesis. Reflecting this, in the absence of lysoPC, serine contributes to 38.2% of PC biosynthesis in the parasite as shown by Wein et al. (2018).

Regarding levels of DAG and TAG in the GDPD-null parasites; in fact we observed increases in DAG but not in TAG levels (only one species of the detected TAG species was significantly upregulated; see Figure 5 —figure supplement 1). We suspect that the disturbance in precursor availability (choline, ethanolamine and serine) reduces DAG usage as the backbone for PL synthesis, resulting in its accumulation. We have now modified the revised manuscript (line 232 onwards) to specifically mention this possibility, as follows: “This disturbance in precursor availability likely reduces usage of DAG, the primary backbone for glycerolipid and neutral lipid production, resulting in its accumulation. Collectively, these results indicated the onset of disruption in PL biosynthesis and were consistent with a major role for PfGDPD in this process.”

In the cell biology study of the KO line, parasites show developmental arrest at trophozoite, reduction in merozoite number as well as effect on food vacuole functioning resulting in decreased haemozoin crystal. These could be generalised stress response and no direct correlation is established with the GDPD reduction. Why haemozoin crystal formation is hindered in the KO parasites, and how is it linked with disturbance in lipid metabolism and specifically PC synthesis?

We agree with the reviewer that the phenotypes described could simply result from a general stress response following GDPD ablation and the resulting decrease in PC levels. In the revised manuscript we have now modified line 326 to reflect this view, as follows: “Ablation of PfGDPD expression produced a phenotypic defect during trophozoite development likely resulting from a direct or general stress response to disrupted PC synthesis, similar to that observed upon inhibition of other enzymes of the CDP-choline pathway (Contet et al., 2015; Gonzalez-Bulnes et al., 2011; Serran-Aguilera et al., 2016).”

We note that Reviewer #3 also raised a similar question regarding haemozoin crystal formation. The reduced haemozoin formation could be a result of the stress response or due to the decrease in TAG levels we observe (original Figure 5C). Neutral lipids have been suggested to play a role in the parasite haem detoxification pathway and promote haemozoin formation (Hoang et al. 2010). Indeed it was recently shown that knockdown of a P. falciparum lysophospholipase results in reduced TAG levels, reduced haemozoin formation and a block in trophozoite development (Asad et al. 2021). It is therefore entirely plausible that the associated developmental defect is due to disruption in haem detoxification rather than directly by reduced PC levels. Since the TAG-haemozoin link is not well established in the current scientific literature (Matz JM et al., 2022), we previously refrained from drawing any further conclusions in our original submission. However, in view of this Reviewer’s comments (as well as those of Reviewer #3 – see below), we now briefly discuss this from line 383 in the revised manuscript – “The reduced haemozoin formation in GDPD-null parasites could also be a result of this decrease in TAG levels. Neutral lipids have been suggested to play a role in the parasite haem detoxification pathway and promote haemozoin formation (Hoang et al., 2010). Indeed it was recently shown that knockdown of a P. falciparum lysophospholipase results in reduced TAG levels, reduced haemozoin formation and a block in trophozoite development (Asad et al., 2021).”

Rescue of KO parasites with choline is intriguing but somewhat inexplicable. In the parasite, majority of PC is synthesised using choline derived from lysoPC, whereas under LPC depleted condition the parasite is shown to utilize PMT pathway for PC synthesis by compensating PE synthesis, rather than utilizing available choline in the serum. Even if some PC gets synthesized using choline taken from milieu, it should be able to recover parasite growth to only a certain level, however, in this study major recovery of parasite growth is shown. Mechanistic proof of choline rescue is missing. It is important to assess phospholipid profile to show normalized PC (and other phospholipids) synthesis under choline rescue condition. The authors should use labelled choline to show its incorporation in the PC synthesised under rescue condition. Can excess glycerophosphocholine (GPC) rescue the parasites under partial knock-down conditions?

It has been shown previously that exogenous choline rescues both wild-type and PMT-null parasites (i.e. lacking a functional SDPM pathway) that otherwise fail to survive (Witola et al., 2008, Wein et al., 2018). The reviewer is correct in observing that choline “should be able to recover parasite growth to only a certain level”. Consistent with that, while we see a significant rescue of parasite growth rate, choline does not completely restore wild-type growth rates (Figure 4B and 4E). This matches previous observations by others (Witola et al., 2008, Wein et al., 2018).

The mechanism underlying the rescue of GDPD-null parasites by choline is important and this question was also raised by Reviewers #2 and #3. A plausible explanation can be gleaned from prior work done in this area. LysoPC is generally the main source of choline for the parasite because at the normal choline concentration in human plasma (~10 uM), choline influx through the RBCM is very low so there is a bottleneck in transport of free choline across the red blood cell membrane (RBCM) (Biagini et al., 2004). However, at supraphysiological concentrations of choline as used in our study (500 uM), choline influx into the infected RBC is increased (Kirk et al., 1991). Incorporation of exogenous choline into PC has been previously demonstrated in lysoPC-starved parasites in Brancucci et al. (2017) using high levels (1mM) of d₉-labelled exogenous choline. Thus, in the absence of lysoPC, the parasite can use exogenous choline to make PC so long as it is provided at relatively high concentrations.

The main question we address in our study is whether disrupting GDPD function mimics a lysoPC-deprived state, thereby implicating GDPD in lysoPC breakdown. We demonstrate this by metabolic labelling using a choline-labelled lysoPC species and show a 25-50% reduction in sourcing of choline from lysoPC in the absence of GDPD. As to the mechanistic proof of choline rescue of GDPD-null parasites, we maintain that we can rely on the previous studies (detailed above) that have reported both choline rescue and choline incorporation into PC in lysoPC-deprived parasites, and that repetition of those data using labelled choline is unnecessary. Instead, in our revised manuscript we have now discussed this issue further (lines 344 onwards), as follows: “Previous work has shown that supplying an excess of choline can rescue the growth of parasites in lysoPC-deprived conditions (Wein et al., 2018; Witola et al., 2008). High concentrations of exogenous choline partially surpass the bottleneck in choline transport across the red blood cell membrane (Biagini et al., 2004) and increase choline influx through infected RBCs (Kirk et al., 1991). LysoPC-deprived parasites can readily take up this choline and use it for PC synthesis as shown by previous metabolic labelling studies (Brancucci et al., 2017). The impact of exogenous choline was pronounced in our PfGDPD-null mutants as they completely failed to survive in the absence of choline but achieved near-wild type growth rates in high choline concentrations. This complete dependence on exogenous choline despite the abundant presence of lysoPC (amounting to ~17% of total lipid content (Garcia-Gonzalo and Izpisua Belmonte 2008)) in the Albumax II-supplemented culture media used in our studies or supplementation with GPC, provides further confirmation that ablating PfGDPD function interferes with choline acquisition from lysoPC and mimics a lysoPC-deprived state.” We trust that these modifications to our manuscript are acceptable to the Reviewer.

Supplying exogenous GPC does not rescue growth of the GDPD-null parasites. We now include these data in the revised submission (Figure 4C) and discuss them in lines 188 and 354.

Other comments:

– I did not get the supplementary figures on the website or in the BioRxiv site.

We trust that this problem is now solved with the submission of our revised manuscript.

– Figure 4B: the parasitaemia is marked in log scale which is confusing to compare between different sets.

We appreciate the Reviewer’s point here but given the wide range of the parasitaemia levels in the -RAP and +RAP cultures, we feel it appropriate to present the data using a log scale to best visualise the differences between the choline-supplemented and control conditions. We hope this is acceptable.

– Figure 5: Why LPCs are getting accumulated in the KO, which are targets of Lysophospholipase, whereas the direct target of GDPD is Glycero-phosphocholine. What is the status of glycero-phosphocholine in these parasites?

The Reviewer raises an interesting point. In our experience, lysoPC measurements vary somewhat in our lipidomics data. This may be due to lysoPC in the Albumax II- supplemented media that may accumulate within the red blood cells (and that we could not get rid of entirely by washing the parasite pellet). As the Reviewer will see from the presented data, the differences in lysoPC levels referred to are not statistically significant due to wide inter-replicate variability. Therefore, we do not attribute much meaning to these “changes” in lysoPC levels.

Regarding the status of GPC in the parasites, this is a polar metabolite and therefore unfortunately was not captured by the lipid extraction and LC-MS methods that we employed in this work.

– Figure 5C: The labelled proportion of the majority of PC species is not significantly different between the two clones.

All species above and including PC(16:0_16:1) show significant reduction in labelled proportions in G1 parasites compared to the B4 controls (a 25-50% decrease with p value <0.05 from three replicates). We had previously shown absolute differences in the dot plot (G1-B4). We have now revised the dot plots shown in Figure 5B and C to present the same data as percentage change in labelling ((G1-B4)/B4 *100) to enable easier interpretation. The changes with p value <0.05 have also been now explicitly highlighted in the revised Figure 5C. Line 258 has also been altered to “However, a consistent and significant decrease (25-50%) in labelling of 10 out of 13 PC species was observed in the choline-starved PfGDPD-null G1 parasites compared to the B4 controls.” We hope these revisions are satisfactory.

– The experimental setup for Figure 5C is confusing, the G1 parasite strain is expected to be completely devoid of PfGDPD, if the GDPD is only enzyme responsible for release of choline from GPC, then how can this parasite utilize lyoPC for labelling the newly synthesised PC.

The reviewer raises an important point here. We also expected no labelling in the G1 parasites, but we suspect that alternate pathways such as direct acylation of lysoPC into PC via the Lands’ cycle could contribute to the labelled fraction in the PC pool. The enzymes involved in these pathways remain to be elucidated. We suggest this in lines 360 onwards of the revised manuscript, as follows: “This suggests that alternate pathways such as (i) direct lysoPC acylation into PC via the Lands’ cycle, (ii) two acylation steps to convert GPC to PC, or (iii) the SDPM pathway may contribute to PC synthesis under choline-starved conditions. However, our results strongly suggest that PfGDPD-mediated choline release from lysoPC remains the primary, indispensable pathway to meet the choline requirements of the intraerythrocytic parasite.” We hope that this text clarifies our interpretation of our observations.

– What is the significance of betaine lipid DGTS upregulation, DGTS is synthesised without the use of choline but using DAGs, how does this data correlate with other lipidomic profile, DAG levels as well as with the choline rescue.

We discuss at length the potential significance of DGTS in the Discussion (lines 389 onwards). Accumulation of DGTS as a non-choline/phosphorous substitute for PC in the choline-starved GDPD-null clonal G1 line is similar to that observed previously in fungi and marine bacteria under similar conditions. As mentioned earlier, due to the presence of residual GDPD levels and possibly activation of the SDPM pathway, RAP-treated cycle 0 GDPD:loxPint:HA schizonts are not severely choline-starved and their lipidomic profile (new Figure 5A) is not relevant here. However, extensive DAG accumulation is seen in both lipidomic profiles due to dysfunctional phospholipid metabolism and likely far exceeds any of its use in DGTS synthesis.

Reviewer #2 (Recommendations for the authors):

1. Is GDPD localization to the PV and/or cytoplasm critical for parasite viability? Some discussion of this question seems warranted at a minimum. This key question could be strongly addressed if the authors extend the experiment in Figure 2G to test if episomal expression of GDPD variants either lacking the SP (and thus presumably just cytoplasmic) or lacking Met19 (which may prevent a cytoplasmic population and just retain the PV population) rescue parasites from loss of endogenous GDPD.

We appreciate the Reviewer’s experimental suggestions here, but respectfully suggest that they address questions that are beyond the remit of the present study. While the suggested experiments appear initially straightforward, they could well turn out to represent a substantial additional workload. The suggestions are based on the reasonable but as yet untested assumption that the internal Met19 is indeed a cryptic translation initiation site. Simple substitution of Met19 might interfere with the enzymatic function of the longer translation product, and different amino acid substitutions may have to be explored. The relative expression levels of the two isoforms (if they exist) may be important. However, we do agree that some discussion of this issue is warranted, and the revised manuscript now includes this on lines 404-411.

2. Do the GDPD mutations studied episomally in Figure 2G impact or reduce protein stability and expression? The microscopy data in the figure supplement suggest that all proteins are expressed but quantitative comparison is difficult. Can the authors show by western blot (normalized to loading control) that WT and enzyme mutants are all expressed at similar levels? This experiment seems critical since the 2012 Klemba study reported that a different active site mutant resulted in insoluble protein.

We accept this point. We have now quantified the mean GDPD-mCherry fluorescence intensity from a number of imaged parasites as a measure of GDPD expression. The results do not detect any statistically significant differences between the different lines. These data have now been added to the revised manuscript as Figure 2—figure supplement 3G. Line 152 of the manuscript has also been amended to read: “Mutagenesis of these key residues did not alter the expression or subcellular localization of the transgenic PfGDPD variants (Figure 2—figure supplement 3F and G) but completely abolished rescue of parasite growth upon disruption of the chromosomal gene (Figure 2G).”

3. Is the mechanism of choline uptake into parasites known? Are New Permeability Pathway mechanisms involved (e.g., does furosemide antagonize uptake?)?

Current understanding of the mechanism of choline uptake is explained above in our response to Reviewer #1. Choline indeed crosses the host red blood cell membrane via the New Permeability Pathway and into the parasite through a parasite choline carrier (Biagini et al., 2004). The revised manuscript now mentions this (line 72 onwards). Choline transport into the host RBC can be blocked by furosemide. However, furosemide does not affect choline uptake by the parasite via the choline carrier.

4. For the experiment in Figure 5A, does addition of exogenous choline to +RAP parasites lead to phospholipid levels that phenocopy -RAP conditions? This experiment may give insight into other GDPD functions beyond choline-dependent phospholipid synthesis.

This is an excellent suggestion and could be considered for future work. We did plan to include a sample corresponding to the G1 clone in the presence of choline for the lipidomics analysis shown in Figure 5C, but due to certain practical restraints we had to limit our analysis to the 2 sample groups shown.

5. The current labeling in Figure 5C is confusing and difficult to match to the relevant textual descriptions, as this same panel is referenced in lines 250 and 259 in different Results sections. The Figure 5C graph on the right (specific PC labeling) is also described in the text before the left graph, which adds confusion. It would be helpful to explicitly label the left graph as total lipids and possibly label the left/right graphs as distinct panels C and D that can be referred to separately and matched with the relevant text descriptions.

We appreciate these suggestions, and in the revised manuscript the figure has now been revised accordingly to make it easier to relate to the relevant text.

6. The 2012 Klemba paper previously showed that recombinant GDPD could hydrolyze glycerophosphocholine. Figure 6 in the present paper is a valuable extension of this experiment to endogenous GDPD from parasites, and these experiments are important to strongly support the proposed enzyme function in choline mobilization. Comparison of IP samples of GDPD {plus minus}RAP or to stable GDPD-deletion clone G1 provides some but incomplete support for specific function, as there could also be other functional losses in parasites that accompany GDPD deletion or co-purifying proteins that accompany GDPD pull-down that impact choline release in these experiments. Do anti-mCherry IPs under +RAP conditions of the parasites complemented with WT but not mutant enzymes (in Figure 2G) phenocopy GPC and choline levels in B4- parasites, as expected if differences between B4- and B4+ or G1 parasites are due to the specific activity of GDPD?

We agree with the reviewer that the earlier demonstration by Denloye et al., 2012 that recombinant GDPD hydrolyses GPC to produce G3P was important, and we were very happy to be able to complement those data in our study by demonstrating that native, parasite-derived enzyme can generate free choline from GPC. Equally importantly, our approach also enabled us to show that even the residual amounts of GDPD in RAP-treated cycle 0 schizonts display measurable catalytic activity, nicely explaining the mild phenotype of the parasites at that point in their development.

We appreciate the Reviewer’s reservations regarding the fact that our pull-down experiments do not completely rule out a role for co-purifying proteins in the GPC hydrolysis we observed. Co-purification of other proteins that contribute to GPC hydrolytic activity is a formal possibility but arguing against this, we did not detect any additional Coomassie-stained bands other than PfGDPD in the SDS PAGE analysis of our pull-down fractions (Figure 6—figure supplement 2). Furthermore, our complementation experiments with WT and mutant mCherry-tagged GDPD show unambiguously that parasite viability is not supported by mutant enzymatically inactive PfGDPD, which might still form interactions with partner proteins. The reviewer’s suggestion is a good one to rule out a possible enzymatic role (or co-factor role) for partner proteins, but we consider this unlikely in the light of knowledge of other GDPD enzymes, which generally function without protein co-factors. From a practical perspective, there is substantial expense associated with the mass-spectrometric analysis required to show hydrolysis to free choline in our assays. For all these reasons, we have not performed the proposed experiments using anti-mCherry to pull-down mCherry-tagged GDPD from parasites complemented with such constructs. We hope this is acceptable.

Reviewer #3 (Recommendations for the authors):

– The catalytic function of this protein has already been investigated in Denloye et al., 2012, this figure should be removed and instead the data from this previous article can be discussed.

It is unclear which figure the reviewer refers to here. If it is Figure 6, we agree with the reviewer that the catalytic function of recombinant GDPD has been partially characterised previously by Denloye et al., 2012. In our revised manuscript we have now added a sentence (line 292) specifically mentioning this, as follows:

“Recombinantly expressed PfGDPD has previously been shown to possess magnesium-dependent hydrolytic activity that releases G3P from GPC (Denloye et al., 2012)”. However, in agreement with the view of Reviewer #2 (see above) we believe it is important to retain our experimental data in the current manuscript for three reasons. First, we confirm the hydrolytic activity of GDPD using a protocol that is completely different from Denloye et al., 2012, in that our in vitro assays used PfGDPD isolated directly from parasites as opposed to the recombinant protein used previously. Second, we used LC-MS to directly quantify GPC and choline in the reactions, whereas Denloye et al., 2012 measured G3P production using a two-step assay as a measure of GDPD activity. Finally – and importantly – our data confirm that even the residual amounts of GDPD in our RAP-treated cycle 0 parasites display measurable, choline releasing catalytic activity. This was crucial to explain the phenotype and lipidomics profile of cycle 0 schizonts. We trust that the reviewer accepts our reasoning on this issue.

– Not all defects, especially at the lipid level, can be explained with the current data and their interpretations: Why Do TAGs and DAGs increase in the mutant? Is there a link between accumulation of hemozoin crystals and neutral lipid accumulation, as recently reported (Asad et al. BMC Biology), have the author tested it? How do the authors explain that choline complementation rescues the parasite when this chline is believed to exclusively being generated from LPC?

We agree with the reviewer that there could indeed be a link between hemozoin generation and neutral lipid accumulation; please see our response above to Reviewer #1. Much further experimentation will be required to confirm this link and we suggest that this is beyond the remit of this current work.

The second point raised by this Reviewer is also mentioned above in our response to Reviewer #1. Briefly, lysoPC is the preferred source of choline for the parasite. However, high concentrations of exogenous choline partially surpass the bottleneck in choline transport across the red blood cell membrane (Biagini et al., 2004) and increase choline influx through the infected RBC (Kirk et al., 1991). LysoPC-deprived parasites readily take up this choline and use it for PC synthesis, as previously shown by metabolic labelling studies (Brancucci et al., 2017).

– Some graphs don't have any error bars.

– Statistical significance is not always shown, and the tests performed are not discussed in the figure captions.

– Representation of the lipidomic data is often hard to follow, individual FA species of each lipid group is only shown in the supplementary figures, which also do not show the statistical significance of the data; were any stat tests done?

– No statistical significance is shown in Figure 5B and C.

– I would propose to the authors to replace the bubble charts that is quite difficult to read and interpret, with only the significant results of PE, PS, PC, LPC and DAG in the form of the bar graphs shown in Figure 5—figure supplement 1.

Statistical significance was calculated for all the data. As stated in the methods, in all cases – “Student’s t-test were used to compare group means and where necessary Bonferroni adjustment for multiple comparisons was applied to the p-value of statistical significance.”

Bar graphs in Figure 5 represent the average labelled proportion in each parasite line. Their variability has been shown as error bars in the dot plots on the side that denote the differences between the proportions. Statistical significance is now shown in the revised Figure 5C for labelling data.

In the main Figure 5C bubble plot, a scale has been provided depicting the p-value associated with each point. Bubbles bigger than the size denoted are statistically significant. In the supplementary figure (Figure 5—figure supplement 1), ONLY the statistically significant species are displayed (as described in the figure legend). We would like to retain the bubble charts as they provide the reader with an eagle-eye’s view of two lipidomic experiments within the space of a single figure and inform on the main changes we discuss in the text. The reader can always then refer to the supplementary bar graphs. We trust this is acceptable.

– In the PfGDPD-null mutant it would of interest to see if the expression of other transcripts has changed, and this can expand on how this parasite can survive solely on the exogenous choline despite having completely lost this proposed essential protein.

– Transcriptional analyses will also reveal why despite the LPC/PC balance being offset, sexual conversion is not induced.

We appreciate these suggestions, and indeed we did initially consider bulk RNAseq analysis of the mutants. However, due to the severe developmental defect we observed in the mutants (in both the 3D7 and NF54 lines), we felt there would be wide transcriptional variations across the board that would likely confound any meaningful changes arising from PfGDPD ablation. Furthermore, since provision of high concentrations of exogenous choline likely merely replaces choline that the parasite would have derived from breakdown of lysoPC, we doubt that there is any alternate pathway to be elucidated through transcriptomics. As described above in our response to Reviewer #2, we suspect that the lack of sexual conversion in the GDPD-null NF54 parasites is simply because the parasites die (due to a choline deficiency) before they can progress to gametocytogenesis.

– The lipidomic analyses is extremely detailed and it seems to be overly condensed to a single image and not fully deliberated in the discussion.

We are glad the reviewer appreciates our lipidomics efforts. Since we also have a large amount of phenotypic and biochemical data (the manuscript already contains 7 figures), we had no choice but to condense the lipidomic work into a themed figure. We have tried to be both informative and concise in our Discussion.

– What is the relation between the accumulation of DGTS and DAG? This could be addressed in the discussion of the paper.

We discuss significance of DGTS in the Discussion (Lines 383-396). While DGTS is made from DAG, a good degree of DAG accumulation is seen in both lipidomic profiles due to dysfunctional phospholipid metabolism and this would far exceed any of its use in DGTS synthesis.

Author details

1. Abhinay Ramaprasad

   Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Contributed equally with

   Paul-Christian Burda

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-9372-5526

2. Paul-Christian Burda

   1. Centre for Structural Systems Biology, Hamburg, Germany
   2. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
   3. University of Hamburg, Hamburg, Germany

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - review and editing

   Contributed equally with

   Abhinay Ramaprasad

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0461-4352

3. Enrica Calvani

   Metabolomics Science Technology Platform, The Francis Crick Institute, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Aaron J Sait

   Electron Microscopy Science Technology Platform, The Francis Crick Institute, London, United Kingdom

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6091-0426

5. Susana Alejandra Palma-Duran

   Metabolomics Science Technology Platform, The Francis Crick Institute, London, United Kingdom

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Chrislaine Withers-Martinez

   Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

7. Fiona Hackett

   Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. James Macrae

   Metabolomics Science Technology Platform, The Francis Crick Institute, London, United Kingdom

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1464-8583

9. Lucy Collinson

   Electron Microscopy Science Technology Platform, The Francis Crick Institute, London, United Kingdom

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

10. Tim Wolf Gilberger

    1. Centre for Structural Systems Biology, Hamburg, Germany
    2. Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
    3. University of Hamburg, Hamburg, Germany

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    For correspondence

    gilberger@bnitm.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7965-8272

11. Michael J Blackman

    1. Malaria Biochemistry Laboratory, The Francis Crick Institute, London, United Kingdom
    2. Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    For correspondence

    Mike.Blackman@crick.ac.uk

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7442-3810

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