Glm S mediated knock-down of a phospholipase expedite alternate pathway to generate phosphocholine required for phosphatidylcholine synthesis in Plasmodium falciparum

Phospholipid synthesis is crucial for membrane proliferation in malaria parasites during the entire cycle in the host cell. The major phospholipid of parasite membranes, phosphatidylcholine (PC), is mainly synthesized through the Kennedy pathway. The phosphocholine required for this synthetic pathway is generated by phosphorylation of choline derived from catabolism of the lyso-phosphatidylcholine (LPC) scavenged from the host milieu. Here we have characterized a Plasmodium falciparum lysophospholipase ( Pf LPL20) which showed enzymatic activity on LPC substrate to generate choline. Using GFP- targeting approach, Pf LPL20 was localized in vesicular structures associated with the neutral lipid storage bodies present juxtaposed to the food-vacuole. The C-terminal tagged glmS mediated inducible knock-down of Pf LPL20 caused transient hindrance in the parasite development, however, the parasites were able to multiply efficiently, suggesting that Pf LPL20 is not essential for the parasite. However, in Pf LPL20 depleted parasites, transcript levels of enzyme of SDPM pathway (Serine Decarboxylase-Phosphoethanolamine Methyltransferase) were altered along with upregulation of phosphocholine and SAM levels; these results show upregulation of alternate pathway to generate the phosphocholine required for PC synthesis through the Kennedy pathway. Our study highlights presence of alternate pathways for lipid homeostasis/membrane-biogenesis in the parasite; these data could be useful to design future therapeutic approaches targeting phospholipid metabolism in the parasite. CSPT, CDP-diacylglycerol-serine-O-phosphatidyltransferase; PSS2, phosphatidylserine synthase by base exchange type 2; PSD, phosphatidylserine decarboxylase, FV, food vacuole; Hb, hemoglobin.


Introduction
Malaria is a major vector-borne infectious disease caused by a protozoan parasite, Plasmodium spp., which is particularly rampant in tropical and subtropical regions of the world. Globally there are more than 229 million cases of malaria worldwide in 2019, and the majority of those, approximately 213 million, were from the African region. This parasitic disease cause ~400,000 deaths globally and the most vulnerable group are the children aged under 5 years [1,2]. In absence of an efficient vaccine for malaria as well as due to the rise of drug-resistant parasite strains, there is an urgent need to identify new drug-targets and development of effective antimalarial. Understanding of key metabolic pathways in the parasite which are essential for parasite survival remains to be the pre-requisite to identify unique and potent targets in the parasites.
The malaria parasite has a complex life cycle in the mosquito vector and human host, having multiple developmental stages. The pathological symptoms are caused by the repeated asexual blood-stage cycle. During the asexual cycle, the parasite replicates rapidly inside the host erythrocyte which requires a vast amount of lipids to support extensive membrane biogenesis for its growth and segregation [3]. These demands can be fulfilled by scavenging the host cell lipid and use them after degradation/modification as well as through de novo synthesis of fatty acids and phospholipids [4][5][6][7]. Phospholipids, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), are the major structural components of the parasite membranes, which have a little amount of cholesterol [8]. In the uninfected erythrocytes, the share of these phospholipids, PC, PE, PS, is 30-40%, 25-35%, and 10-20%, respectively; however, parasites/infected erythrocytes contain 20-55% of PC, 15-40% of PE and 4-15% PS [9,10].
The phosphatidylcholine is a major membrane phospholipid, which can be synthesized in Plasmodium parasites by two routes including the cytidine diphosphate-or CDP-choline Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200549/914990/bcj-2020-0549.pdf by guest on 22 June 2021 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200549 branch of the Kennedy pathway and the Serine Decarboxylase-Phosphoethanolamine Methyltransferase (SDPM) pathway. The Kennedy pathway is responsible for 89% of PC synthesis in the parasite using choline; the choline first gets converted into phosphocholine by the action of choline kinase and then enters in the Kennedy pathway [11]. The SDPM pathway involves five enzymes that uses host serine and fatty acids as precursors for PC synthesis. There are three sequential methylation steps of phosphoethanolamine by phosphoethanolamine methyltransferase (PfPMT) produce phosphocholine, which then enters in the Kennedy pathway [12][13][14]. It has been shown that PC synthesis uses choline generated from the catabolism of lysophosphatidylcholine scavenged in the host milieu [9].
A recent study suggested that lysophosphatidylcholine (LPC) is essential to maintain the asexual cycle, whereas in LPC depleted conditions the parasites switch to the sexual commitment [15]. To obtain the phospholipid building blocks, complex phospholipids obtained from the host are to be catabolized by the action of hydrolases including phospholipases and lysophospholipases [16][17][18] putative LPLs, out of which ten LPLs have high sequence similarity (36-61%) with each other; these proteins are ~400 amino acid long without having any signal sequences [19].
The other four LPLs are 675-921 amino acid long and have a signal peptide. The characteristic GXSXG motif is conserved among all lysophospholipases wherein, the active site residue serine (S) is flanked by the glycine. The catalytic triad is composed of serine (S), aspartate (D), and histidine (H). Several studies have characterized parasite phospholipases Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200549/914990/bcj-2020-0549.pdf by guest on 22 June 2021 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200549 5 in the malaria parasite for their role in asexual and gametocytic stages of the parasites [20,21]. One of the most characterized α/β hydrolases is an esterase, PfPARE (PF3D7_0709700), although this gene is not essential at the asexual blood stage of the parasite, but its expression changes in pepstatin-resistant parasites [22]. Another Plasmodium protein with phospholipase activity was found to play role in hepatocytic egression [23]. Conditional knock-down of PfPAPTPL1 (Patatin like phospholipase 1) showed that it is dispensable for the asexual staged but PfPAPTPL1 depleted gametocytes have reduced flagellation and egress. In contrast to this, delayed gametocyte maturation has been observed in PNPLA1/PATPL depleted parasites instead of the defect in flagellation [20,21]. LPLs are also expected to play role in the catabolism of host-derived complex lipids, such as LPC, to obtain building blocks for parasite phospholipid synthesis. However, there are very few studies for the localization and functional significance of parasite lysophospholipases.
Here, we report the biochemical and functional characterization of one of the P. falciparum LPL, named PfLPL20 (PlasmoDB gene ID: PF3D7_0702200). The recombinant PfLPL20 was expressed, purified and its LPL specific enzymatic activity was confirmed.
We used the GFP-glmS ribozyme system for localization and conditional knockdown of the native protein in the parasite. The protein was found to be associated with the neutral lipid storing bodies in the asexual blood-stage parasites; efficient growth of parasites under conditional knockdown of PfLPL20 confirms its dispensability for parasite survival.
However, alteration in the SDPM pathway genes expression and accumulation of phosphocholine as well as S-adenosylmethionine in the PfLPL20 depleted parasites, indicated a switch to the SDPM pathway to generate the phosphocholine required for the Kennedy pathway-dependent synthesis of PC.

Parasite culture, plasmid construct, and parasite transfection
Plasmodium falciparum strain 3D7 was cultured in RPMI media (Invitrogen) supplemented with 0.5% (w/v) Albumax (Invitrogen) 4% haematocrit. The culture was kept static in a gas mixture of 5% carbon dioxide, 5% oxygen, and 90% nitrogen at 37°C. To generate an overexpression line, the full-length pfLPL20 gene was amplified using primers 1294A, 1295A and cloned into pSSPF2-GFP vector. Parasite cultures were synchronized by repeated sorbitol treatment and 100 g of purified plasmid DNA (Plasmid Midi Kit, Qiagen, Valencia, CA) was transfected in P. falciparum by electroporation (310 V, 950 F) [43].
Transfected parasites were selected over 2.5 μg/ml blasticidin (Calbiochem). To generate PfLPL20-glmS-GFP construct, C-terminal fragment (750 bp long) of pfLPL20 gene was amplified using specific primers 1224A and 1225A and cloned into glmS-GFP vector [44]. The C-terminal fragment was cloned in-frame to the N-terminus of GFP in the SpeI and KpnI restriction enzymes sites. Transfected parasites were selected over 2.5 μg/ml blasticidin (Calbiochem) and subsequently subjected to on and off cycling of blasticidin drug to promote the integration of the plasmid in the main genome. Integration was confirmed by PCR using 2200F and 1234A and by western blotting also by using an anti-GFP antibody. Individual parasite clones were isolated by serial dilution method, and integration was confirmed in the clonal parasite population.

Conditional knock-down analysis and in-vitro growth assays
To assess the effect of knock-down of PfLPL20 on the parasite, PfLPL20-glmS-GFP transgenic parasites were tightly synchronized with 5% sorbitol and grown with media containing 2.5mM concentration of glucosamine (GlcN) (Sigma-Aldrich) or solvent alone (incomplete media, iRPMI). For microscopic analysis and morphology of the parasites, Giemsa-stained smears were prepared at every 8hr interval from both glucosamine treated and control parasites. Parasite growth was assessed after 48 and 96 hours by flow cytometry using BD FACS Calibur (Beckton Dickinson). Briefly, cells were incubated with EtBr for 30 minutes at 37°C in dark followed by two washing with 1×PBS. These samples were analyzed by fluorescence counting, 100,000 events were acquired using BD FACS Calibur system (Beckton Dickinson); EtBr-stained uninfected erythrocytes were used as background control. To assess the effect of glucosamine on the down regulation of PfLPL20 at the protein level, PfLPL20-GFP-glmS ring stage parasites were treated with 2.5 mM glucosamine, harvested at the schizont stage, and subjected to the western blotting.

Parasite fractionation and Western blotting
Parasites were harvested at the trophozoite stage by lysis of infected RBCs using 0.15% saponin; the supernatant was collected separately, and the parasites pellet was lysed by the freeze-thaw cycle. Laemmli buffer was added to both the fractions and proteins were separated in 12% SDS-PAGE. Subsequently, fractionated proteins were transferred to PVDF membrane (Millipore) and incubated with blocking buffer (4% skim milk or 3% BSA in 1× PBS). Blots were incubated with primary antibody (rabbit anti-GFP 1:10000, rabbit anti-BiP 1:15000), washed 5 times with 1×PBS, and probed with HRP conjugated respective secondary antibody (1:100000). Subsequently, blots were washed with 1×PBS and visualized using a thermo-scientific ECL kit.

Immuno-fluorescence assay (IFA) and fluorescent microscopy
Infected RBCs were fixed in 4% paraformaldehyde and 0.05 % glutaraldehyde. After permeabilization with 0.1 % TritonX-100, the cells were incubated with rabbit polyclonal anti-GFP (1:500) for 2 hours and subsequently labelled with Alexa Flour-488. Labeled 8 parasites were washed three times with 1×PBS. Parasite nuclei were stained with DAPI at a final concentration of 5 µg/ml. To label the neutral lipid storage structures, parasites were stained with Nile Red (Molecular Probes) using a modified method [34]. Briefly, Nile Red was added to a final concentration of 1 µg/ml into the parasite culture (5-10% parasitemia), the culture was incubated on ice for 30 min and subsequently washed with 1×PBS before analysis by confocal microscopy. Images were captured by Nikon A1 confocal laser scanning microscope and analysed by Nikon-NIS element software (version 4.1). The 3D images were constructed by using series of Z-stack images using IMARIS 7.0 (Bitplane Scientific) software.  supplementary Table S1)], and 1× SYBR Green PCR mix. The threshold cycles (CTs) generated by qPCR system were used to calculate fold change for relative quantitative analysis. Each assay was carried out in triplicate, and the experiments were repeated thrice. Parasites were isolated from Infected red blood cells (5×10 7 cell equivalents) from both PfLPL20-iKD and control by using saponin lysis. Their total lipids were extracted by chloroform: methanol, 1:2 (v/v) and chloroform: methanol, 2:1 (v/v) as previously described [4]. The standard compounds (choline, phosphocholine, and S-adenosylmethionine) were purchased from Sigma-Aldrich in form of choline chloride, phosphorylcholine chloride calcium salt tetrahydrate, and S-(5′-Adenosyl)-L-methionine p-toluene sulfonate salt and were dissolved in HPLC grade solvents. Samples were analyzed by 3200-QTRAP LC-MS system for estimation as described previously [45].

Expression, and purification of recombinant protein
Full-length pfLPL20 gene was amplified by using specific primers 2200F and 2200R and cloned into pETM41 vector between NcoI and XhoI sites to give pETM41-His6-MBP-PfLPL20 construct. The recombinant protein PfLPL20 was expressed as a soluble protein in the cytosol of the E. coli BL21(DE3) codon + cells. For expression, bacterial cells having pETM41-His6-MBP-PfLPL20 were grown in LB medium supplemented with kanamycin at 37°C. Expression of the fusion protein was induced using 1mM isopropyl β-Dthiogalactoside (IPTG) overnight at 16°C. The next day, cells were harvested and resuspended into lysis buffer (25mM Tris, pH7.4, 500mM NaCl, 10mM Imidazole) and lysed by sonication (Vibra cell sonicator). The supernatant was subjected to a combination of Ni-NTA affinity chromatography followed by amylose affinity chromatography. Eluted fractions were subjected to SDS-PAGE to assess the purity of the recombinant protein.

Lysophospholipase (LPL) activity assay using LPC as substrate and enzyme kinetics
For assessing the enzymatic activity of the recombinant PfLPL20, a lysophospholipase (LPL) activity assay was established using lysophosphatidylcholine as substrate. The activity

Statistical Analysis
The data sets were analysed using GraphPad Prism ver 5.0 to calculate Km value and the datasets were compared using unpaired Student's t-test.

PfLPL20 is an active lysophospholipase having an alpha/beta hydrolase domain
PfLPL20 is 424 amino acid long 49.9 kDa protein having putative transmembrane region (37 to 55 amino acids) and two hydrolase domains (82-151 aa and 194 -386 aa; Pfam PF12146 E-value 2.3e -05 and 2.2e -14 respectively) ( Figure 1A). Although both the domains contain characteristic hydrolase motif GXSXG, the three active site residues (S218, D349, H375) are present only in the second hydrolase domain (Supplementary figure 1). The active site S218 residue in the conserved motif is located in the characteristic 'nucleophile elbow' of hydrolase domains [24]. PfLPL20 is annotated as a putative lysophospholipase in the Plasmodium genome database, PlasmoDB. To ascertain and characterize the enzymatic activity of PfLPL20, recombinant protein corresponding to full-length gene was expressed using E. coli expression system and purified by using a combination of affinity chromatography techniques. The purified recombinant-PfLPL20 migrated as ~90kDa protein on SDS-PAGE, which is slightly above the expected size of ~84kDa ( Figure 1B).
The purified recombinant protein was used in a fluorescence-based activity assay of lysophospholipase using LPC substrate ( Figure 1C). The recombinant PfLPL20 showed concentration dependent activity in the assay, confirming it to be a lysophospholipase. The Km value enzyme was found to be 33.41µM and the R 2 value for the kinetics was found to be 0.955. (Figure 1D).

Stage specific subcellular localization of PfLPL20
To study the localization of PfLPL20 in asexual stage parasites, we generated the transgenic parasite line for episomal expression of PfLPL20-GFP fusion protein. Expression of the fusion protein was confirmed by western blotting (Figure 2A), and these transgenic parasites were studied for localization of the PfLPL20-GFP fusion protein by fluorescence/confocal We tagged the endogenous PfLPL20 to study its functional essentiality by conditional knock-down strategy. We used GFP-glmS ribozyme system for C-terminal tagging of the native pfLPL20 gene by single cross-over, so that the fusion protein gets expressed under the control of the native promoter ( Figure 3A). A transgenic parasite line, PfLPL20-GFP-glmS, was obtained and individual parasite clones were isolated; C-terminal integration was confirmed using PCR-based analyses ( Figure 3B). Expression of the fusion protein of ~70kDa was detected by western blot analysis using anti-GFP antibody ( Figure 3C); this band was not detected in wild type 3D7 parasites. Further, western blot analysis for different parasite developmental stages, showed expression at all the stages, with trophozoites having the maximum expression levels ( Figure 3D). Localization of endogenously tagged PfLPL20-GFP protein showed similar localization as in the case of episomally expressed protein. The GFP signal in these parasites was observed in vesicular structures in the parasite cytosol as distinct foci/vesicle during trophozoite stages, whereas during late-trophozoite and schizont stages, the labelling was observed in large vesicular structure present juxtaposed to the foodvacuole ( Figure 3E). Further, staining with Nile red showed that these vesicular structures are neutral lipid-rich body; Nile red staining showed overlap with the endogenously tagged PfLPL20-GFP protein in these structures ( Figure 3F).

PfLPL20 is dispensable at the asexual stage of the parasite
To understand the functional significance of PfLPL20 and its possible involvement in lipid metabolism, we utilized the transgenic PfLPL20-GFP-glmS parasite line for glmS ribozyme mediated inducible knock-down of PfLPL20 expression. Inducible knock-down (iKD) of pfLPL20 was assessed by estimating corresponding mRNA levels as well as levels of the PfLPL20-GFP levels in the PfLPL20-iKD set ( Figure 4A, B). To assess the effect of this PfLPL20 knock-down on parasite growth, synchronized parasite cultures were grown in the presence or absence of glucosamine, and new ring-stage parasites were counted for two subsequent cell-cycles using flow-cytometry as well as using Giemsa stained smears of parasite cultures. However, we could not observe any change in the parasite growth in PfLPL20-iKD set as compared to controls ( Figure 4D), which may indicate the dispensability of this gene for the asexual blood-stage parasite.
Further, to study the effect of selective degradation of PfLPL20 on the parasite development cycle, Giemsa-stained smears of the parasites were prepared at different time points during the assay. In the control set, during each intra-erythrocytic cycle, the parasites developed from ring to trophozoites to mature schizonts, and subsequently, merozoites released from these schizonts invaded new erythrocytes, which effectively increased the total parasitemia. In PfLPL20-iKD set, the parasites developed from ring to trophozoites stages, however, several trophozoites showed an interruption in further development ( Figure 4E). It was observed that this growth interruption was only transient, which led to a slight delay in parasite development, however, all the parasites were able to develop into mature stages and overall parasite growth and multiplication was similar as in the case of the control set. As described above, the PfLPL20 was localized in a neutral lipid body present juxtaposed to the food-vacuole, therefore, we assessed the effect of PfLPL20 knockdown on these lipid bodies.
However, there was no change in the size or Nile Red staining of neutral lipid bodies in the PfLPL20-iKD parasites ( Figure S3). shown to influence phosphatidylcholine synthesis pathways in the parasite through induction of SDMP pathway genes and shift towards using Serine or Ethanolamine as preferential substrate. Since PfLPL20 is involved in cleavage LPC, hence its knock-down may deplete the production of choline substrate required for PC synthesis and thus mimic LPC depleted conditions; therefore, we assessed if this influence the expression level of genes of PCbiosynthesis as suggested by Brancucci et al. Real-time quantitative PCR based analyses was carried out for genes pfSAMS, pfPMT, pfEK as well as for pfLPL20. In addition, we also assessed transcript levels of choline kinase, pfCK, which is also plays role in phosphocholine generation in the PC synthesis pathway. As described above, there was a significant reduction (>60%) of the pfLPL20 in the PfLPL20-iKD set as compared to the control set; whereas, pfSAMS expression level was significantly increased ~3.5 fold in the PfLPL20-iKD set. There was no change in the expression levels of pfPMT, pfEK, and pfCK ( Figure 5A).
The upregulation of pfSAMS in PfLPL20-iKD conditions suggests that SDPM pathway may have switched higher synthesis of choline-phosphate, which could be utilized for PC-synthesis by the Kennedy pathway. We compared the levels of choline, phosphocholine, and SAM in PfLPL20-iKD set as compared to the control set. The LC-MS analysis showed that there was no significant change in the choline levels for the two sets ( Figure 5B), however, phosphocholine levels were significantly high in PfLPL20-iKD set as compared to control ( Figure 5C), in addition, SAM levels were also found to be upregulated in PfLPL20-iKD set as compared to control ( Figure 5D). This data correlated with the upregulation of SDPM pathway in the knock-down conditions. The asexual growth cycle of the malaria parasite involves an extensive synthesis of lipids, especially those required for the development of new membranes. Indeed, there is a huge increase in phospholipids, neutral lipids, and fatty acids content in the parasite as it develops inside the host erythrocyte [25]. Several studies showed that the metabolic pathways linked with synthesis, catabolism, and trafficking of lipids, are essential for parasite survival [17,[26][27][28]. The parasite can carry out de novo synthesis as well as scavenge lipids/phospholipids from the host milieu during the erythrocytic cycle, which are metabolized and used for its growth.

Discussion
In our attempts to further understand lipid homeostasis and membrane synthesis in the parasite, we explored the role of a putative lysophospholipase (LPL) in P. falciparum. In P. falciparum-infected erythrocytes, lysophospholipase-like activity was found to be increased up to 3-fold [29], suggesting functional relevance of this enzyme for parasite growth and segregation. We have carried out the biochemical and functional characterization of putative a lysophospholipase, labelled as PfLPL20 (Gene ID: PF3D7_0702200). The P.
falciparum genome codes for a large family of about 14 LPLs, however, their functional roles are not characterized for many of the LPLs. All the LPLs harbors α/β hydrolase domain having GXSXG conserved motif. PfLPL20 also contains this conserved motif having serine, histidine, and aspartic acid as catalytic site residues, conserved for all the lysophospholipases across kingdoms. The recombinant PfLPL20 showed LPL specific activity utilizing LPC as substrate. Indeed, it is shown that the LPC scavenged from the host milieu is used to generate metabolites to be utilized by the parasite for its growth [9]. Enormous research has been carried out to develop different reverse genetic approaches for gene function analysis in malaria parasites [30]. As it is difficult to knockout an essential gene in the parasite, several tools have been developed for transient knockdown of the target gene in the parasite [31,32]. We used the glmS ribozyme system for conditional knock-down of the target gene; we tagged PfLPL20 at C-terminal with GFP-glmS tag, so that the transgenic parasites can be studied for both localization and gene function analysis. In addition, a transgenic parasite line expressing PfLPL20-GFP fusion protein was also generated for localization studies. Confocal microscopic analysis of both transgenic parasite lines (PfLPL20-GFP and PfLPL20-GFP-glmS) showed that PfLPL20 is present in vesicular structures, which are associated with parasite membrane or distributed in the parasite cytosol; in late stages, these vesicles fused in a large vesicular structure close to the food-vacuole. These structures showed resemblance with the previously reported neutral lipid bodies (NLB) [33,34], which act as a reservoir of diacylglycerols (DAG) and triacylglycerol (TAG). Co-localization of PfLPL20 with neutral lipid markers confirmed its presence in these lipid bodies. It is suggested that the parasite utilizes these stored lipids for membrane biogenesis during segregation. Localization of PfLPL20 in small endocytic vesicles near parasitophorous vacuole and its subsequent localization in neutral lipid stores may indicate its involvement in the catabolism of lipids that are scavenged from the host. Indeed, it has been shown that P. falciparum scavenges LPC from the host milieu which gets catabolized and utilized in PC synthesis required for membrane biogenesis [9]. As expected from sequence homology and presence of the catalytic active site, PfLPL20 showed catabolic activity on LPC substrate, which may suggest its role in phospholipid recycling in the parasite. Overall, the localization studies and biochemical characterization of PfLPL20 indicate its association with lipid homeostasis in the parasite.
The C-terminal glmS tag-based system could induce >60% knock-down of pfLPL20 transcript in the transgenic parasites; whereas the parasite growth analysis showed that the inducible-knock-down did not affect the parasite growth and multiplication, which may suggest that the enzyme is not essential for the survival of the asexual blood-stage parasites.
A recent study using piggyBac transposon insertion-based mutagenesis study also showed the dispensability of PfLPL20 [35]. PfLPL20 shows a high degree of similarity with another hydrolase (PfPARE), which is also not essential at the asexual stage but mutations in this gene are responsible for resistance to new potential drugs [22]. One of the reasons for dispensability of an active enzyme in the cell could be compensation of its function by any other enzymes/pathway. Biochemical characterization shows that catabolism of LPC is the main function of PfLPL20 which generates fatty acids and choline to be utilized by the parasite. Although the parasite can import free choline from the host milieu [36,37], however, the scavenged choline is not utilized by the parasite for membrane synthesis [9].
Detailed analysis of parasite stage development in the PfLPL20 knockdown suggested transient morphological effects on trophozoite stages. These results may suggest the possibility that the PfLPL20 function is compensated or switched over to another metabolic pathway in the parasite. P. falciparum harbours more than ten LPLs, and it is possible that any other LPL takes over the function of PfLPL20 under its knockout conditions. Further, the knockdown by glmS strategy is not absolute, it is also possible that the partial knockdown is not able to completely down-regulate the PfLPL20 protein in the cell, and the remaining amount of PfLPL20 is sufficient to carry out its required function for cell survival.
Extensive synthesis of membrane phospholipids is essential for parasite growth and segregation. Trophozoite and schizont infected erythrocytes show a six-fold increase in the phospholipid content in comparison to uninfected erythrocytes; most of this phospholipid content is phosphatidylcholine (PC), which is a major membrane phospholipid in Plasmodium. Under normal conditions, the parasite generated most of the required PC via CDP-Choline-dependent-Kennedy pathway using choline as a substrate [38][39][40][41]. Recent studies have shown that LPC imported from the serum is the main source of choline which converts into phosphocholine to get utilized for PC biosynthesis [9,15], thus lysophospholipases play a key role in the membrane biosynthesis. The parasite can also Downloaded from http://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200549/914990/bcj-2020-0549.pdf by guest on 22 June 2021 utilize triple methylation of the phosphoethanolamine (PE) to make phosphocholine which then enters in the same pathway to make PC ( Figure 6). This involves the activity of phosphoethanolamine methyltransferase (PMT), which transfers a methyl group from Sadenosylmethionine (SAM) to PE. Further, under serum LPC depleted conditions, there is a shift of PC synthesis towards the PMT pathway using ethanolamine substrate [9]. It was also shown that under LPC depleted conditions the expression of PfPMT is also shown that in field-isolates high plasma LPC has a negative influence on gametocyte production [42], it was suggested that the LPC depletion acts upstream of any event of sexual differentiation, but its exact role in induction of sexual commitment remains unclear.
In addition, quantitative evaluation of metabolites showed upregulation of phosphocholine as well as SAM levels in the PfLPL20 knock-down conditions. This shows that upregulation of SAMS under PfLPL20-iKD conditions resulted in increased conversion of L-methionine to SAM, resulting in the accumulation of SAM and thus its higher availability for triple methylation step by PfPMT; consequently, the PMT pathway got upregulated to produce higher levels of phosphocholine. These results thus confirm that under PfLPL20 knockdown conditions, the alternate pathway gets upregulated to provide phosphocholine required for PC biosynthesis, which helps asexual stage parasites to survive and multiply. However, it is also possible that some other LPL in the parasite takes over the function to certain extents, to compensate the loss of PfLPL20. We would also like to point out that knockdown of the PfLPL20 protein in this study was not absolute, and therefore the remaining amount of PfLPL20 may still carry out its functional role in the cell.
Overall, our results indicate PfLPL20 plays a role in PC biosynthesis through catabolism of LPC acquired from host milieu to provide choline which gets phosphorylated to form the phosphocholine; under the PfLPL20 depleted conditions, the parasite switches an alternate SDPM pathway for phosphocholine synthesis, which is utilized for PC biosynthesis. Thus, the parasite can compensate for the loss of one pathway of phosphocholine synthesis with another pathway, so that the parasite growth and segregation are being maintained. These results may suggest that PfLPL20 levels regulate cooperation between two different pathways to generate phosphocholine required for PC synthesis.
These data provide new information on membrane phospholipid biosynthetic pathways of the malaria parasite, which could be used to design future therapeutic approaches.