A choline-releasing glycerophosphodiesterase essential for phosphatidylcholine biosynthesis and blood stage development in the malaria parasite
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eLife assessment
This high-quality study characterizes a key enzyme in asexual red blood stages of the malaria parasites that is used to salvage lipid precursors needed for membrane biogenesis and parasite growth in red blood cells. A previously identified glycerophosphodiesterase (PfGDPD), is shown to mediate the hydrolysis of host lyso-phosphatidycholine to generate choline, which in turn is required for parasite de novo phosphatidylcholine synthesis. Extensive analysis of the localization, growth phenotype and lipidomic profiles of PfGDPD deficient parasites indicate that this salvage pathway is essential for lipid homeostasis and asexual parasite development.
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Abstract
The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here, we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Genetic ablation of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.
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Author Response
Reviewer #1 (Public Review):
In this manuscript by Ramaprasad et al., the authors report on the functional characterization of the P. falciparum glycerophosphodiesterase to assess its role in phospholipid biosynthesis and development of asexual stages of the parasite. The authors utilized loxP strategy to conditionally knock-out the target gene, they also carried out complementation assays to show recovery of the knock-out parasites. They further show that Choline supplementation is also able to rescue the knock-out phenotype. Quantitative lipidomic analyses show effect on majority of membrane phospholipids. In vitro activity assays and metabolic labelling assays shows role of GDPD in metabolism of exogenous lysoPC for PC synthesis. The manuscript deciphers the functional role of an important component of lipid …
Author Response
Reviewer #1 (Public Review):
In this manuscript by Ramaprasad et al., the authors report on the functional characterization of the P. falciparum glycerophosphodiesterase to assess its role in phospholipid biosynthesis and development of asexual stages of the parasite. The authors utilized loxP strategy to conditionally knock-out the target gene, they also carried out complementation assays to show recovery of the knock-out parasites. They further show that Choline supplementation is also able to rescue the knock-out phenotype. Quantitative lipidomic analyses show effect on majority of membrane phospholipids. In vitro activity assays and metabolic labelling assays shows role of GDPD in metabolism of exogenous lysoPC for PC synthesis. The manuscript deciphers the functional role of an important component of lipid metabolism and phospholipid synthesis in the parasite, which are crucial metabolic pathways required for replication of the parasite in the host cell.
We thank the Reviewer for assessing our work and for the following helpful suggestions.
Reviewer #2 (Public Review):
The authors use a conditional Lox/Cre knock-out system to test and confirm the essentiality of glycerophosphodiester phosphodiesterase (GDPD) for blood-stage parasites and a key role in mobilizing choline from precursor lysophosphocholine (LPC) for parasite phospholipid synthesis. Prior works had identified serum LPC as the key choline source for parasites, localized this enzyme in parasites, and suggested an essential function in releasing choline, but this key function had remained untested in parasites. This manuscript critically advances mechanistic understanding of parasite phospholipid metabolism and its essentiality for blood-stage Plasmodium and identifies a potential new drug target.
Overall, this study is well constructed and rigorously performed, and the data provide strong support for the central conclusions about GDPD essentiality and functional contribution to parasite phosphocholine metabolism. The observation that exogenous choline largely rescues parasites from lethal deletion of GDPD is especially compelling evidence for a critical and dominant role in choline mobilization. A few aspects of the paper, however, are not fully supported by the current data and/or need clarification.
We thank the reviewer for this very positive assessment and the helpful suggestions below.
- GDPD localization
a) The authors conclude that GDPD is localized to the parasitophorous vacuole (PV) and parasite cytoplasm (lines 114-115), which is consistent with the prior 2012 Klemba paper. However, the data in the present paper (Figures 2A and 2E) only seem to support cytoplasmic localization but don’t obviously suggest a population in the PV, in part because no co-staining with a PV marker is shown. The legend for Fig. 2E indicates staining with the PV marker, SERA5, but such co-stain is not shown in the figures or figure supplements. This data should ideally be included and described.
We apologise for this error and omission in our original submission. In response to this suggestion, we have now generated new data that demonstrate co-localisation of the PV marker SP-mScarlet (Mesen-Ramirez et al., 2019) with GDPD in our GDPD-GFP line. In the revised manuscript we now include those new data in Fig 2A and we have also corrected the legend of the revised Fig 2E to reflect what is being shown.
b) How do the authors explain cytoplasmic localization for GDPD? This protein contains an N-terminal signal peptide, which can account for secretion to the PV but would contradict a cytoplasmic population. The 2012 Klemba paper suggested that internal Met19 might provide an alternate site for translation initiation without a signal peptide and thus result in cytoplasmic localization. Some discussion of this ambiguity, its relation to understanding GDPD function, and a possible path to resolve experimentally seem necessary, especially as the authors suggest from data in Fig. 7 that this enzyme may have functions beyond choline mobilization, which may relate to distinct forms in different sub-cellular compartments.
The Reviewer raises an excellent point here. We agree that the apparent dual localization of GDPD and the question of its potential function in both compartments is intriguing. Since lysoPC is efficiently internalised into the parasite, one simple possible explanation (which we failed to state earlier) is that GDPD performs a similar enzymatic function in both compartments. Given the importance of choline for parasite membrane biogenesis, it would not be surprising for GDPD activity to be required at high abundance in order to maintain sufficient choline levels in the parasite. We have now modified lines 403 onwards in the revised Discussion to provide more perspective on this point, as follows: “Based on protein localisation, ligand docking and sequence homology analyses, we can further speculate regarding aspects of PfGDPD function 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 also do not rule out catalytic activity towards additional glycerophosphodiester substrates such as glycerophosphoethanolamine and glycerophosphoserine (Figure 6-figure supplement 1A and 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.”
- The phenotypes depicted by representative microscopy images in panel 4E (especially for choline rescue) should be supported by population-level analysis by flow cytometry or microscopy of many parasites to establish generality.
We agree that this would be informative, and in the revised manuscript we have now added a representative microscopy image as source data (Figure 4E_G1+Cho48h-sourcedata.png). It is also worth pointing out that G1 is a clonal line generated from the RAP+ Choline+ parasite population. Both population-level analysis by flow cytometry (Fig 4A) and microscopic images (Fig 4D) are therefore also applicable to the G1 line.
- The analysis in the last results section (starting on line 296) seems preliminary.
a) For panel 7B, a population analysis of many parasites, with appropriate statistics, is important to establish a generalizable defect beyond the single image currently provided.
b) The data here would seem to be equally explained by an alternative model that GDPD∆ parasites are competent to form gametocytes but their developmental stall (due to choline deficiency) prevents progression to gametocytogenesis. The authors speculate that GDPD may play other roles in phospholipid metabolism beyond choline mobilization that are essential for gametocytogenesis. Their model, if correct, predicts that a GDPD deletion clone from +RAP treatment that is rescued by exogenous choline should not form gametocytes. Testing this prediction would be important to strongly support the conclusion of broader roles for GDPD in sexual development beyond choline mobilization.
We interpreted our results precisely as the reviewer suggests here – that the developmental stall during trophozoite stages is severe enough to prevent sexual differentiation. A priori, we have no reason to suspect that GDPD plays other roles that are selectively essential for gametocyte development. We speculated that GDPD might have other roles in asexual stages but not necessarily based on this experiment. In the revised manuscript we have modified line 313 accordingly to remove ambiguity: “This result implies that the loss of PfGDPD causes a severe block in PC synthesis resulting in the death of asexual parasites before they get to form gametocytes.”
We have also altered line 411 in the Discussion to: “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.”
We agree with the reviewer that the analysis is preliminary. Since we lose RAP-treated GDPD:HA:loxPintNF54 populations after cycle 1, we were unable to do more detailed analysis with the line. We also wished to carry out the experiment that the reviewer suggests here to analyze choline-rescued mutants. However, we would be unable to test for this as choline supply alone would suppress sexual differentiation in these parasites (as shown in Brancucci et al., 2017).
Reviewer #3 (Public Review):
In this work, Ramaprasad et al. aimed to investigate the roles of a glycerophosphodiesterase (PfGDPD) in blood stage malaria parasites. to determine its role, they generated a conditional disruption parasites line of PfGDPD using the DiCre system. RAP-induced DiCre-mediated excision results in removal of the catalytic domain of this protein. Loss of this domain leads to a significant reduction of parasite survival, specifically affecting trophozoite stages. They suggest that there is an invasion defect when this protein domain is deleted. They additionally show the introduction of an episomal expression of PfGDPD can rescue the activity of the protein and restore parasite development. Interestingly, exogenous choline can rescue the effects of the loss of PfGDPD. Lipidomic analyses with labelled LPC show that choline release from LPC is severely reduced upon protein ablation and in turn prevents de novo PC synthesis. These experiments also show increase in DAG levels suggesting a defect in the Kennedy pathway. The authors purified PfGDPD and enzymatically show that this protein facilitates the release of choline from GPC. Additionally, the paper also briefly looks at the effects of the protein during sexual blood stages and show this is unlikely to be involved in sexual differentiation.
This paper is of interest to the community since the breakthrough paper of Brancucci et al. (2017), which showed us that decreased LPC levels induce sexual differentiation. This work brings novel insight into a GDPD responsible for the release of choline from GPC which actual seems more relevant to asexual stages and not sexual stage parasites. The authors have been extremely thorough in their experimentations on parasite viability and the exact role of this protein.
We thank the reviewer for this positive assessment and the helpful comments.
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eLife assessment
This high-quality study characterizes a key enzyme in asexual red blood stages of the malaria parasites that is used to salvage lipid precursors needed for membrane biogenesis and parasite growth in red blood cells. A previously identified glycerophosphodiesterase (PfGDPD), is shown to mediate the hydrolysis of host lyso-phosphatidycholine to generate choline, which in turn is required for parasite de novo phosphatidylcholine synthesis. Extensive analysis of the localization, growth phenotype and lipidomic profiles of PfGDPD deficient parasites indicate that this salvage pathway is essential for lipid homeostasis and asexual parasite development.
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Reviewer #1 (Public Review):
In this manuscript by Ramaprasad et al., the authors report on the functional characterization of the P. falciparum glycerophosphodiesterase to assess its role in phospholipid biosynthesis and development of asexual stages of the parasite. The authors utilized loxP strategy to conditionally knock-out the target gene, they also carried out complementation assays to show recovery of the knock-out parasites. They further show that Choline supplementation is also able to rescue the knock-out phenotype. Quantitative lipidomic analyses show effect on majority of membrane phospholipids. In vitro activity assays and metabolic labelling assays shows role of GDPD in metabolism of exogenous lysoPC for PC synthesis. The manuscript deciphers the functional role of an important component of lipid metabolism and …
Reviewer #1 (Public Review):
In this manuscript by Ramaprasad et al., the authors report on the functional characterization of the P. falciparum glycerophosphodiesterase to assess its role in phospholipid biosynthesis and development of asexual stages of the parasite. The authors utilized loxP strategy to conditionally knock-out the target gene, they also carried out complementation assays to show recovery of the knock-out parasites. They further show that Choline supplementation is also able to rescue the knock-out phenotype. Quantitative lipidomic analyses show effect on majority of membrane phospholipids. In vitro activity assays and metabolic labelling assays shows role of GDPD in metabolism of exogenous lysoPC for PC synthesis. The manuscript deciphers the functional role of an important component of lipid metabolism and phospholipid synthesis in the parasite, which are crucial metabolic pathways required for replication of the parasite in the host cell.
-
Reviewer #2 (Public Review):
The authors use a conditional Lox/Cre knock-out system to test and confirm the essentiality of glycerophosphodiester phosphodiesterase (GDPD) for blood-stage parasites and a key role in mobilizing choline from precursor lysophosphocholine (LPC) for parasite phospholipid synthesis. Prior works had identified serum LPC as the key choline source for parasites, localized this enzyme in parasites, and suggested an essential function in releasing choline, but this key function had remained untested in parasites. This manuscript critically advances mechanistic understanding of parasite phospholipid metabolism and its essentiality for blood-stage Plasmodium and identifies a potential new drug target.
Overall, this study is well constructed and rigorously performed, and the data provide strong support for the central …
Reviewer #2 (Public Review):
The authors use a conditional Lox/Cre knock-out system to test and confirm the essentiality of glycerophosphodiester phosphodiesterase (GDPD) for blood-stage parasites and a key role in mobilizing choline from precursor lysophosphocholine (LPC) for parasite phospholipid synthesis. Prior works had identified serum LPC as the key choline source for parasites, localized this enzyme in parasites, and suggested an essential function in releasing choline, but this key function had remained untested in parasites. This manuscript critically advances mechanistic understanding of parasite phospholipid metabolism and its essentiality for blood-stage Plasmodium and identifies a potential new drug target.
Overall, this study is well constructed and rigorously performed, and the data provide strong support for the central conclusions about GDPD essentiality and functional contribution to parasite phosphocholine metabolism. The observation that exogenous choline largely rescues parasites from lethal deletion of GDPD is especially compelling evidence for a critical and dominant role in choline mobilization. A few aspects of the paper, however, are not fully supported by the current data and/or need clarification.
1. GDPD localization
a) The authors conclude that GDPD is localized to the parasitophorous vacuole (PV) and parasite cytoplasm (lines 114-115), which is consistent with the prior 2012 Klemba paper. However, the data in the present paper (Figures 2A and 2E) only seem to support cytoplasmic localization but don't obviously suggest a population in the PV, in part because no co-staining with a PV marker is shown. The legend for Fig. 2E indicates staining with the PV marker, SERA5, but such co-stain is not shown in the figures or figure supplements. This data should ideally be included and described.b) How do the authors explain cytoplasmic localization for GDPD? This protein contains an N-terminal signal peptide, which can account for secretion to the PV but would contradict a cytoplasmic population. The 2012 Klemba paper suggested that internal Met19 might provide an alternate site for translation initiation without a signal peptide and thus result in cytoplasmic localization. Some discussion of this ambiguity, its relation to understanding GDPD function, and a possible path to resolve experimentally seem necessary, especially as the authors suggest from data in Fig. 7 that this enzyme may have functions beyond choline mobilization, which may relate to distinct forms in different sub-cellular compartments.
2. The phenotypes depicted by representative microscopy images in panel 4E (especially for choline rescue) should be supported by population-level analysis by flow cytometry or microscopy of many parasites to establish generality.
3. The analysis in the last results section (starting on line 296) seems preliminary.
a) For panel 7B, a population analysis of many parasites, with appropriate statistics, is important to establish a generalizable defect beyond the single image currently provided.b) The data here would seem to be equally explained by an alternative model that GDPD∆ parasites are competent to form gametocytes but their developmental stall (due to choline deficiency) prevents progression to gametocytogenesis. The authors speculate that GDPD may play other roles in phospholipid metabolism beyond choline mobilization that are essential for gametocytogenesis. Their model, if correct, predicts that a GDPD deletion clone from +RAP treatment that is rescued by exogenous choline should not form gametocytes. Testing this prediction would be important to strongly support the conclusion of broader roles for GDPD in sexual development beyond choline mobilization.
-
Reviewer #3 (Public Review):
In this work, Ramaprasad et al. aimed to investigate the roles of a glycerophosphodiesterase (PfGDPD) in blood stage malaria parasites. to determine its role, they generated a conditional disruption parasites line of PfGDPD using the DiCre system. RAP-induced DiCre-mediated excision results in removal of the catalytic domain of this protein. Loss of this domain leads to a significant reduction of parasite survival, specifically affecting trophozoite stages. They suggest that there is an invasion defect when this protein domain is deleted. They additionally show the introduction of an episomal expression of PfGDPD can rescue the activity of the protein and restore parasite development. Interestingly, exogenous choline can rescue the effects of the loss of PfGPDP. Lipidomic analyses with labelled LPC show that …
Reviewer #3 (Public Review):
In this work, Ramaprasad et al. aimed to investigate the roles of a glycerophosphodiesterase (PfGDPD) in blood stage malaria parasites. to determine its role, they generated a conditional disruption parasites line of PfGDPD using the DiCre system. RAP-induced DiCre-mediated excision results in removal of the catalytic domain of this protein. Loss of this domain leads to a significant reduction of parasite survival, specifically affecting trophozoite stages. They suggest that there is an invasion defect when this protein domain is deleted. They additionally show the introduction of an episomal expression of PfGDPD can rescue the activity of the protein and restore parasite development. Interestingly, exogenous choline can rescue the effects of the loss of PfGPDP. Lipidomic analyses with labelled LPC show that choline release from LPC is severely reduced upon protein ablation and in turn prevents de novo PC synthesis. These experiments also show increase in DAG levels suggesting a defect in the Kennedy pathway. The authors purified PfGDPD and enzymatically show that this protein facilitates the release of choline from GPC. Additionally, the paper also briefly looks at the effects of the protein during sexual blood stages and show this is unlikely to be involved in sexual differentiation.
This paper is of interest to the community since the breakthrough paper of Brancucci et al. (2017), which showed us that decreased LPC levels induce sexual differentiation. This work brings novel insight into a GDPD responsible for the release of choline from GPC which actual seems more relevant to asexual stages and not sexual stage parasites. The authors have been extremely thorough in their experimentations on parasite viability and the exact role of this protein.
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