TgSEC14-LTP1 is a vacuole-secreted SEC14/CRAL-TRIO-like protein required for host DAG flux and PC homeostasis in T. gondii

SUMMARY

Intracellular apicomplexan parasites depend on host-derived lipids to expand the parasitophorous vacuole (PV) and replicate. Toxoplasma gondii secretes numerous dense granule proteins (GRAs) for PV remodeling, but PV lumen factors that selectively bind host lipids and shape host-to-parasite lipid flux remain poorly defined. Here, we identify TgSEC14-LTP1, a previously uncharacterized GRA with a divergent SEC14/CRAL-TRIO domain. Structural and genetic analyses show that TgSEC14-LTP1 is secreted into the PV lumen and is essential for parasite replication. Lipidomic profiling, protein-lipid pulldown assays, ¹³C-labeling-based lipid flux analysis, and fluorescent lipid uptake assays show that TgSEC14-LTP1 binds phosphatidylcholine (PC) and diacylglycerol (DAG) and is required for efficient host-to-parasite PC and DAG flux and parasite lipid homeostasis. TgSEC14-LTP1 depletion reduces parasite PC and DAG pools, disrupts bulk PC synthesis and parasite biogenesis, delays egress, and ultimately impairs growth. These findings identify TgSEC14-LTP1 as a key Sec14-like PV lumen factor that links lipid binding to host lipid acquisition and PC homeostasis, underscoring the importance of host lipid scavenging for parasite survival.

In brief

Cruz-Miron et al. identify TgSEC14-LTP1, a SEC14/CRAL-TRIO-like protein in the parasitophorous vacuole lumen with a hydrophobic-lipid-binding cavity. Conditional depletion reveals its essential role in parasite growth by regulating host-to-parasite diacylglycerol and phosphatidylcholine flux and maintaining lipid homeostasis, leading to reduced parasite size, defective cytokinesis, and delayed egress.

Graphical Abstract

INTRODUCTION

Toxoplasma gondii is an apicomplexan parasite that can infect a wide range of warm-blooded animals, including humans, in whom it can cause severe disease, particularly in immunocompromised individuals. An estimated one-third of the world’s population is chronically infected with Toxoplasma, making it the most prevalent parasitic zoonosis worldwide.^1,2 During the acute phase of infection, the tachyzoite stage of the parasite proliferates actively within host cells, residing in a specialized compartment known as the parasitophorous vacuole (PV).³ Acquisition of host-derived nutrients is crucial at this stage, as Toxoplasma lacks the enzymatic machinery required for de novo biosynthesis of purines, tryptophan, arginine, and cholesterol.⁴ To compensate for these metabolic deficiencies, the parasite employs salvage pathways to acquire additional amino acids, lipids, and pyrimidines from the host cell.^5–7

Numerous Toxoplasma secreted proteins, many of which involved in the acquisition of host-derived nutrients, are secreted into the PV from specialized secretory organelles known as dense granules.^4,8,9 These secreted proteins, collectively referred to as GRAs, number approximately 124 based on hyperplexed localization of organelle proteins by isotope tagging (hyperLOPIT) analyses; yet fewer than half have been functionally characterized.¹⁰ Several GRAs, including GRA23, GRA17,¹¹ GRA47,^12,13 and GRA72, are thought to form pores in the PV membrane (PVM), thereby permitting the passage of small molecules ranging from 1,300 to 1,900 Da.¹² These permeation processes facilitate parasite growth and contribute to the maintenance of ion homeostasis within the PV.

Host-derived lipid acquisition is essential for Toxoplasma growth and development as it sustains membrane biogenesis and maintenance throughout the parasite’s lytic cycle.^14,15 Toxoplasma constantly scavenges host lipids, which are remodeled and channeled to the neutral lipid synthesis pathway to be stored in parasite lipid droplets and mobilized to support parasite division and prevent lipotoxic death.^14–17 Multiple cellular mechanisms that the parasite uses to obtain host lipids have been described, including recruitment of host organelles,^15,18,19 formation and sequestration of giant multivesicular bodies,²⁰ and the activity of GRA proteins that remodel host structures. For example, GRA7 promotes the formation of host-organelle-sequestering tubules (HOST), which facilitate the transport of organelles such as lysosomes and lipid droplets into the PV lumen.^21,22 The parasite takes up complex phospholipids, such as phosphatidylcholine (PC), and remodels or recycles them to obtain key building blocks required for its own bulk phospholipid synthesis.²³ Within the PV, TgLCAT, an enzyme with phospholipase A2 (PLA2) and cholesterol trans-acyltransferase activities, degrades scavenged phospholipids into lysophospholipids and fatty acids (FAs), while esterifying cholesterol with FAs that are subsequently transported and incorporated into phospholipid synthesis pathways.²³ Recently, GRA38, a dense granule phosphatidic acid phosphatase (PAP) resident in the PV lumen, was shown to regulate the phosphatidic acid-diacylglycerol balance and parasite fitness under lipid-rich conditions.²⁴ This shows that, as in Plasmodium falciparum, the parasite scavenges complex lipids, which parasite enzymes such as PAPs, phospholipases A, and lipases hydrolyze into key precursors that contribute to bulk phospholipid synthesis and, consequently, parasite growth and survival.^25–29 Additional mechanisms include ceramide uptake via the micropore³⁰ and the activity of ATP-binding cassette (ABC) transporters, which may facilitate the transport of cholesterol and phospholipids across the parasite plasma membrane.³¹ Despite these advances, the molecular mechanisms by which lipids are transported across the PV lumen to the parasite remain poorly understood.

While vesicular trafficking can contribute to membrane remodeling, lipid transport often occurs through non-vesicular mechanisms that enable rapid redistribution of lipids while preserving membrane integrity.^32–34 A major class of proteins mediating non-vesicular lipid trafficking is the lipid transfer proteins (LTPs),³⁵ which contain hydrophobic pockets that bind specific lipid species and can promote lipid exchange between membranes or regulate lipid metabolism in vivo.^34,35 Among LTPs, the SEC14/CRAL-TRIO superfamily is particularly well characterized for its role in lipid exchange. SEC14-domain-containing proteins feature a hydrophobic cavity formed by three α-helices and five β-sheets, along with a gate-like structure that dynamically regulates lipid entry and exit.^36–38 In Plasmodium falciparum, SEC14-domain-containing proteins have been shown in vitro to transfer cholesterol and specific phospholipids between membrane compartments.^39,40 Although Toxoplasma is predicted to encode 15 SEC14-domain-containing proteins, their functions remain largely unexplored, raising the possibility that some may contribute to lipid transport within the PV.³⁹

Here, we identified and functionally characterized TGGT1_215160, a previously unstudied GRA protein annotated as a hypothetical protein with no assigned function. Structural homology analysis using Foldseek, applied to the AlphaFold-predicted tertiary structure, shows that TGGT1_215160 contains a SEC14-like domain featuring a hydrophobic cavity consistent with lipid-binding activity, despite the absence of identifiable homology at the primary sequence level. Our results show that TGGT1_215160 localizes to the PV lumen, binds diacylglycerol (DAG) and PC, and is required for efficient host-derived DAG and PC flux into parasite lipid pools that support bulk phospholipid synthesis and normal parasite growth and survival. Disruption of TGGT1_215160 alters parasite lipid homeostasis and reduces parasite PC levels. We therefore renamed TGGT1_215160 as TgSEC14-LTP1, a PV lumen SEC14-domain lipid-binding protein required for efficient host-derived DAG flux and PC homeostasis during intracellular growth.

RESULTS

TgSEC14-LTP1 is a vacuole-secreted SEC14-domain protein essential for tachyzoite growth

To identify GRA proteins that contribute to host lipid acquisition and parasite lipid homeostasis in Toxoplasma, we focused on those that are essential for parasite survival. Although several known GRA proteins (e.g., GRA2, GRA3, GRA6, and GRA7) contain lipid interaction motifs,^21,41 none are indispensable for parasite viability, indicating they are unlikely to account for an essential lipid-dependent function during the tachyzoite lytic cycle.

To refine our search, we leveraged hyperLOPIT-based subcellular data, which identified approximately 124 GRA proteins.¹⁰ Cross-referencing this dataset with genome-wide CRISPR-Cas9 fitness profiling⁴² revealed 11 GRA-encoding genes with fitness scores below −4.5, a threshold indicative of essentiality during the tachyzoite lytic cycle. Notably, eight of these candidates were annotated as hypothetical proteins lacking functional annotation. To infer potential functional activity, we conducted structure-based similarity screening using Foldseek,⁴³ applying it to the AlphaFold-predicted tertiary structures of these eight hypothetical GRAs. Foldseek alignment against the Protein Data Bank (PDB) revealed that two candidates, ToxoDB: TGGT1_215160 and ToxoDB: TGGT1_228760, share structural homology to the SEC14/CRAL-TRIO domain—a lipid-binding fold characterized by a hydrophobic cavity that can support lipid exchange. Based on this domain prediction, we provisionally designated these proteins as TgSEC14-LTP1 and TgSEC14-LTP2, respectively. Subsequent phylogenetic analysis confirmed that TgSEC14-LTP1 clusters with SEC14-domain-containing orthologs from Rattus norvegicus and Homo sapiens (Figure 1A). Pairwise alignment revealed that TgSEC14-LTP1 shares ~10.4% sequence identity with the human SEC14-like protein and ~8.5% identity with its R. norvegicus counterpart (Figure S1A), consistent with conservation of the overall fold despite substantial primary sequence divergence (Figure S1A).

Figure 1. Conditional depletion of TgSEC14-LTP1 blocks the lytic cycle of Toxoplasma tachyzoites.

(A) Neighbor-joining phylogenetic tree showing the relationship between TgSEC14-LTP1 and structurally homologous SEC14-domain-containing proteins, with bootstrap values based on 500 trials.

(B) CRISPR/Cas9-mediated 3^′-HA-tagging for TgS EC14-LTP1 using a dimerizable Cre recombinase (DiCre) system. The pU6-Cas9-Tg215160 sgRNA construct (encoding Cas9 and TgSEC14-LTP1--specific sgRNA) was transfected into the RHΔku80-DiCre strain alongside a donor amplicon containing HA-FLAG-LoxP-Sag1-DHFR-HXGPRT, flanked by 40 bp 5^′/3^′-homology arms. Hypoxanthine- and mycophenolic-acid-resistant tachyzoites expressing HXGPRT were cloned and screened by PCR. The resulting Tg215160-DiCre strain (iKDTgS EC14-LTP1) enabled conditional knockdown of TgS EC14-LTP1 upon Rapa treatment.

(C) Immunofluorescent images showing TgS EC14-LTP1 localization in the DiCre mutant at 24 and 48 h with or without 50 nM Rapa. Scale bar: 5 μm.

(D) Immunoblot confirming expression of TgS EC14-LTP1-HA in the transgenic strain and its depletion following Rapa treatment.

(E) Crystal-violet-stained plaques illustrating parasite lytic cycles in confluent HFF monolayers (blue).

(F) Quantification of plaque numbers per well (n = 4 trials; significance was tested by one-way ANOVA; mean ± SE; ****p ≤ 0.0001).

To investigate the function of TgSEC14-LTP1, we generated a conditional knockdown strain (iKDTg215160-DiCre), hereafter referred to as iKDTgSEC14-LTP1, using the U1-snRNP gene silencing strategy.⁴⁴ In this approach, the endogenous TgSEC14-LTP1 locus (TGGT1_215160) was modified at its 3^′ end by inserting a DiCre-responsive cassette containing homology arms, a C-terminal HA epitope tag, a hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) resistance cassette, and two LoxP recombination sites (Figure 1B). This system enables Cre-recombinase-dependent transcript degradation upon treatment with rapamycin (Rapa), thereby allowing controlled silencing of TgSEC14-LTP1. Rapa treatment induces recombination at the introduced LoxP sites, leading to mRNA degradation (Figure 1B). CRISPR/Cas9-mediated homologous recombination was used to introduce the cassette in-frame at the C terminus of TgSEC14-LTP1, ensuring expression of a tagged fusion protein under endogenous regulatory control (Figure 1B). Clonal parasites were selected with hypoxanthine and mycophenolic acid, followed by limiting dilution and PCR confirmation (Figure S1B). Immunofluorescence analysis of the resulting iKDTgSEC14-LTP1 strain confirmed that the tagged TgSEC14-LTP1-HA protein localizes to the parasitophorous vacuole lumen (Figure 1C), consistent with its identity as a secreted dense granule protein. Upon Rapa induction, TgSEC14-LTP1-HA levels were markedly reduced, as demonstrated by both immunofluorescence and immunoblotting after 48 h (Figures 1C and 1D), validating the efficiency of conditional gene silencing at the protein level.

To determine whether TgSEC14-LTP1 is essential for the parasite’s lytic cycle, we performed plaque assays by infecting human foreskin fibroblast (HFF) monolayers with either wild-type (WT) or iKDTgSEC14-LTP1 parasites in the presence or absence of Rapa. After 7 days, iKDTgSEC14-LTP1 parasites treated with Rapa failed to form plaques, whereas plaques were observed for the WT parental strain (−/+ Rapa) and for iKDTgSEC14-LTP1 without Rapa (Figure 1E and 1F). These phenotypic results, in combination with structural and phylogenetic analyses demonstrating homology of TgSEC14-LTP1 to SEC14-domain proteins, confirm that TgSEC14-LTP1 is an essential effector required for parasite propagation. The DiCre-inducible knockdown system thus functionally validates TgSEC14-LTP1 as a critical mediator of Toxoplasma lytic cycle progression.

To further validate the essential role of TgSEC14-LTP1, we generated an independent conditional knockout strain using the DiCre recombinase system (iKOTgSEC14-LTP1), which enables complete excision of the TgSEC14-LTP1 locus following Rapa treatment. Immunofluorescence and immunoblot analyses confirmed the loss of TgSEC14-LTP1 in DiCre-excised parasites, while plaque assays revealed a comparable defect in the parasite lytic cycle to that observed in the U1-snRNP knockdown strain (Figures S2A–S2E). Notably, the DiCre knockout system exhibited an additional phenotype, characterized by incomplete tachyzoite division, in which daughter cells remained partially connected at the posterior end, suggesting a role for TgSEC14-LTP1 in facilitating proper cytokinesis (Figure S2C). Complementation of iKOTgSEC14-LTP1 with a TgSEC14-LTP1-Myc allele restored parasite growth, as assessed by plaque assay and immunofluorescence (Figures S2G and S2H), further supporting the role of TgSEC14-LTP1 in maintaining parasite fitness.

TgSEC14-LTP1 is a dense granule protein that is important for parasite growth and egress

To determine whether TgSEC14-LTP1 functions as a dense granule protein, we examined its subcellular localization in tachyzoites by immunofluorescence assays (IFAs). In intracellular parasites, TgSEC14-LTP1-HA signal overlapped with GRA2, a well-characterized marker of dense granules, confirming its classification as a GRA and its secretion into the PV lumen (Figures 2A, 2B, and S2C). To assess the dynamics of protein depletion upon conditional silencing, we treated iKDTgSEC14-LTP1 parasites with Rapa and monitored TgSEC14-LTP1 expression over time. At 24 h post-treatment, TgSEC14-LTP1-HA persisted within dense granules (Figure 2A), whereas by 48 h, the signal was largely abolished, indicating efficient degradation of the targeted transcript and loss of protein expression (Figure 2B). Similarly, TgSEC14-LTP1-HA levels became undetectable by western blot at 48 h post-Rapa treatment within the iKO system (Figure S2D). Additionally, we observed that TgSEC14-LTP1 accumulated in distinct foci near the periphery of the PVM and within the center of the PV (Figure 2C, white arrows), suggesting its association with the intravacuolar network (IVN).

Figure 2. TgSEC14-LTP1 localizes to dense granules in Toxoplasma, and its deletion impairs parasite growth and egress.

(A) Immunofluorescence showing co-localization of TgSEC14-LTP1 with GRA2 in intracellular tachyzoites 24 h post-infection (−/+ Rapa). Parasites were stained with α-HA and α-GRA2 antibodies. Scale bar: 5 μm.

(B) Immunofluorescence images of extracellular tachyzoites harvested 48 h post-infection (−/+ Rapa), labeled with α-HA and α-GRA2 antibodies. Differential interference contrast (DIC) and DAPI staining indicate parasite morphology and nuclei. Scale bar: 2 μm.

(C) Confocal (left) and STED (right) microscopy images showing the association of TgSEC14-LTP1 with the IVN. Orthogonal views and white arrows indicate foci of TgSEC14-LTP1 accumulation corresponding to the IVN. Scale bar: 2 μm.

(D) Replication efficiency of iKDTgSEC14-LTP1 and parental strains at 24 and 48 h post-infection (−/+ Rapa). Intracellular tachyzoites were quantified based on SAG1 staining (parasites per vacuole) to assess proliferation rates.

(E) Endodyogeny analysis, showing the formation of daughter cells within a parent cell, assessed by IMC1-positive daughter cells in tachyzoites.

(F) Induced egress of parasites after 48 h of reinfection (− /+ Rapa treatment), measured following ionophore (1 μM A23187) treatment.

(G) Invasion efficiency of the indicated strains under −/+ Rapa conditions. Bar graphs represent data from 100 to 200 parasites (n = 3–4 assays; significance was tested by two-way ANOVA; mean ± SE; **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

To evaluate the functional consequences of TgSEC14-LTP1 depletion on the parasite lytic cycle, we quantified replication, invasion, and egress in the iKD and iKO TgSEC14-LTP1 strain following Rapa treatment. At both 24 and 48 h post-induction, Rapa-treated parasites formed significantly fewer large vacuoles compared to untreated controls, indicating a defect in intracellular replication (Figures 2D and S2E). Endodyogeny assays (Figure 2E) showed a marked reduction of 45%–47%, compared to control, in the number of daughter cell formation per tachyzoite upon TgSEC14-LTP1 knockdown, consistent with impaired cell division. Because cellular stress can promote stage conversion toward bradyzoites, we assessed the expression of the cyst wall marker CST1. Approximately 10% of vacuoles in TgSEC14-LTP1-deficient parasites were CST1 positive (Figures S3A and S3B), consistent with a modest stress response rather than wholesale stage conversion.

To assess egress efficiency, parasites were treated with the calcium ionophore A23187,⁴⁵ which showed that 10% of TgSEC14-LTP1-deficient parasites exhibited delayed or incomplete egress relative to controls (Figure 2F). In contrast, no significant differences were observed in invasion efficiency between Rapa-treated and untreated iKDTgSEC14-LTP1 parasites (Figure 2G), indicating that TgSEC14-LTP1 is dispensable for host cell entry but critical for intracellular growth and important for timely egress.

To evaluate whether depletion of TgSEC14-LTP1 affects parasite morphology and vacuolar architecture, we performed IFAs using GRA23 as a marker of the PVM and IMC1 to delineate the parasite inner membrane complex (Figure S3C). Quantitative analysis showed that PVs containing four or eight parasites were significantly smaller, by 28% and 37% respectively, in TgSEC14-LTP1-depleted parasites compared to controls (Figure S3D), suggesting impaired vacuole expansion during intracellular replication. Individual parasites within the PV were also smaller, with a 27% reduction in length and a 19% reduction in width upon TgSEC14-LTP1 knockdown (Figures S3E and S3F), indicating a defect in parasite growth and morphogenesis.

Together, these data establish TgSEC14-LTP1 as a secreted dense granule protein required for normal parasite growth and development, PV expansion, and vacuolar organization, thereby supporting efficient replication and timely egress during the tachyzoite lytic cycle.

TgSEC14-LTP1 is dispensable for PVM integrity

Given its localization to the PV lumen, TgSEC14-LTP1 was hypothesized to contribute to PV lumen processes that support vacuole expansion during intracellular division. To determine whether TgSEC14-LTP1 contributes to maintaining PVM integrity, we examined the localization of GRA1, a dense granule protein typically retained within the PV, in wild-type and TgSEC14-LTP1-depleted parasites (Figure S2F). Mislocalization of vacuolar GRAs into the host cell cytoplasm has previously been used as a marker of PVM rupture.⁴⁶ Immunofluorescence analysis demonstrated that, even after 4 days of infection, GRA1 remained confined within the PV in TgSEC14-LTP1-deficient parasites, with no detectable cytoplasmic diffusion (Figure S2F). It is noteworthy that in the absence of TgSEC14-LTP1, GRA1 staining intensity is increased compared to untreated parasites (Figure S2F), suggesting that protein synthesis is not impaired and that the parasites remain metabolically active.

To further assess whether the MYR1-dependent effector translocation machinery remains functional in the absence of TgSEC14-LTP1, we monitored the export of GRA16, a canonical MYR1-dependent effector, to the host cell nucleus. Immunofluorescence analysis revealed that GRA16 nuclear accumulation was not negatively affected in the TgSEC14-LTP1-deficient parasites compared to the wild-type strain (Figure S2I), indicating that the MYR1 translocon remains operational despite TgSEC14-LTP1 depletion.

These results indicate that PVM integrity and MYR1-dependent effector export are not compromised upon TgSEC14-LTP1 depletion. We therefore favor a model in which TgSEC14-LTP1 supports parasite growth through PV lumen functions that influence lipid homeostasis rather than through structural maintenance of the PVM.

Lipid droplet formation in Toxoplasma is reduced in the absence of TgSEC14-LTP1

Recent studies have shown that scavenged lipids are channeled into parasite lipid droplets (LDs), which are then mobilized during replication to support daughter cell formation.^15,47,48 To determine whether TgSEC14-LTP1 contributes to this process, we assessed LD abundance following TgSEC14-LTP1 depletion. Host cells were infected with tachyzoites for 24 h, fixed, and stained with BODIPY 493/503 to label neutral lipid stores (Figures 3A–3C). Quantitative analysis showed that intracellular TgSEC14-LTP1-deficient parasites had an approximately 80% reduction in LD abundance compared with WT and untreated mutant controls (Figures 3B and 3D). A 38% reduction in LD abundance was also observed in released TgSEC14-LTP1-depleted parasites (Figures 3C and 3D), consistent with a broad defect in neutral-lipid storage.

Figure 3. The conditional deletion of TgSEC14-LTP1 impairs the formation of lipid droplets and lipid acquisition in the parasite.

(A) Workflow of lipid droplet staining in intracellular and released parasites.

(B) Immunofluorescence assay showing LDs labeled with BODIPY 493/503 in the cytoplasm of intracellular parasites. Scale bar: 5 μm.

(C) LDs staining in released parasites (−/+ Rapa). Scale bar: 2 μm.

(D) Quantification of the ratio of LDs found a single parasite, in intracellular and released parasites (−/+ Rapa), with or without OA supplementation. An average of 100 parasites per condition was analyzed (n = 3; significance was tested by unpaired t test; mean ± SE; *p ≤ 0.05 and**p ≤ 0.01). (E) Schematic representation of the experimental strategy used for lipid uptake analysis in intracellular parasites by IFA. HFFs were incubated with OA for 24 h, washed, and then incubated with BODIPY 493/503 for 1 h. After thorough washing, cells were incubated for an additional 3 h before infection with WT and iKDTgSEC14-LTP1 strains (−/+ Rapa).

(F) Infected cultures processed for IFA 24 h post-infection. Scale bar: 5 μm.

(G) Quantification of BODIPY 493/503 fluorescence in WT + Rapa and iKDTgSEC14-LTP1 −/+ Rapa parasites, measured by microscopy (n = 4; significance was tested by one-way ANOVA; mean ± SE; ****p ≤ 0.0001).

Oleic acid (OA) increases host neutral-lipid stores and enriches 18:1-containing phospholipids.^47–49 In our assays, OA increased parasite LD abundance even upon TgSEC14-LTP1 depletion, indicating that neutral-lipid droplet accumulation is not completely blocked in the absence of TgSEC14-LTP1. However, OA supplementation did not restore parasite growth (Figures S4A and S4B), arguing that the essential function of TgSEC14-LTP1 is not explained solely by reduced neutral-lipid storage. The higher LD counts in the released parasite group likely reflect improved visualization of isolated parasites with reduced host background and overlap, rather than additional OA exposure (Figures 3B and 3C). Taken together, these data indicate that TgSEC14-LTP1 promotes normal LD biogenesis in Toxoplasma and supports the conclusion that TgSEC14-LTP1 function is required for lipid homeostasis needed for replication, beyond neutral-lipid storage.

TgSEC14-LTP1 promotes accumulation of host-prelabeled neutral lipids in intracellular parasites

Given that TgSEC14-LTP1 depletion impairs LD accumulation in Toxoplasma, we asked whether TgSEC14-LTP1 is required for efficient accumulation of host-prelabeled neutral lipid signal within parasite LDs. To this end, HFFs were treated with OA-albumin for 24 h to induce LD accumulation, followed by extensive washing and staining with BODIPY 493/503 to label host neutral-lipid pools. After a subsequent 3-hour incubation to allow redistribution of the labeled lipid signal, OA-treated HFFs were infected with WT or iKDTgSEC14-LTP1 parasites in the presence or absence of Rapa (see diagram, Figure 3E). Fluorescence microscopy revealed a reduced BODIPY-positive LD signal within TgSEC14-LTP1-depleted parasites compared with both WT + Rapa and untreated iKDTgSEC14-LTP1 controls (Figures 3F and 3G). Quantification of BODIPY-positive LDs per parasite confirmed an 84% decrease in LD signal in the TgSEC14-LTP1 knockdown condition (Figures 3F and 3G).

LDs consist primarily of neutral lipids, mainly triacylglycerols and cholesteryl esters, but their biogenesis requires polar phospholipids, including PC, which forms the LD surface monolayer and contributes substrates for neutral lipid synthesis. Together, these results indicate that TgSEC14-LTP1 is required for efficient LD loading from host neutral lipid pools under OA-driven conditions. This phenotype is consistent with a broader role for TgSEC14-LTP1 in parasite lipid homeostasis and motivated our lipidomic analysis below.

The absence of TgSEC14-LTP1 disrupts PC homeostasis in Toxoplasma

To assess global effects of TgSEC14-LTP1 depletion, we performed LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) lipidomic analyses on TgSEC14-LTP1-depleted and TgSEC14-LTP1-expressing strains. GC-MS profiling revealed a broad disruption of fatty acid (FA) content and homeostasis (Figure 4A) and a reduction of long-chain polyunsaturated species, particularly arachidonic acid (C20:4), which the parasite must scavenge from the host.^20,50,51 To define the lipid class level changes associated with TgSEC14-LTP1 loss, we performed LC-MS/MS lipidomics in both the iKO and iKD parasite lines (Figures 4B and S5). TgSEC14-LTP1 depletion resulted in a pronounced remodeling of the parasite lipidome. More particularly, lipidomics revealed a 39% decrease in PC relative abundance, along with reduced levels of lysophosphatidylcholine (LPC), free FA (FFA), and DAG, which collectively contribute substrates for de novo synthesis of PC. TgSEC14-LTP1 depletion also reduced PI and cholesterol abundance (Figure 4B). In contrast, sphingomyelin (SM), lysophosphatidylethanolamine (LPE), and phosphatidylethanolamine (PE) increased, consistent with compensatory phospholipid remodeling previously observed in Plasmodium under PC limitation.²⁹

Figure 4. TgSEC14-LTP1 deficiency impairs host-derived phospholipid acquisition.

(A) Effects of iKOTgSEC14-LTP1 on FA profile (mol%) of intracellularly growing parasites. Parasites were either left untreated (ut) or treated with 50 nM Rapa for 24 or 48 h. FAs predicted to be synthesized de novo by the parasite via the prokaryotic type II FA synthase (FASII) pathway in the apicoplast or scavenged from the host cell are indicated below the graph. A bar graph showing a magnified view of the quantity (mol%) of scavenged FAs in the iKOTgSEC14-LTP1 strain is shown above (n = 4; significance was tested by Student’s t test).

(B) Relative abundance ratios of the indicated lipids. Data represent the mean ± standard deviation (SD) of biological replicates (n = 4).

(C) Schematic workflow of stable isotope labeling of host cells to assess the contribution of TgSEC14-LTP1 to host lipid scavenging.

(D) Abundance of host-derived ¹³C-labeled phospholipids (n = 4). Significance was tested by two-way ANOVA followed by Sidak’s multiple comparisons test are indicated. Data are presented as mean values +/− SEM (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Further molecular profiling of lipid species in both the iKD and iKO strains revealed a consistent reduction in PC, LPC, DAG, and FFA species enriched for unsaturated acyl chains commonly associated with host-derived lipid pools, including C18:1, C18:2, and C20:4 (Figures S6A and S6B). In the iKO strain (Figure S6B), PC species such as PC 16:0_18.1 were reduced (by ~20%), together with corresponding DAG and FFA species (e.g., DAG 16:0_18:1, FFA 18:1, and FFA 20:4). Multiple LPC species (including LPC 16:0, LPC 18:1, and LPC 20:4) were also diminished, consistent with altered PC turnover and lipid remodeling. The iKD dataset (Figure S6A) supported these findings, with significant reductions in PC species such as PC 18:1_18:1, PC 16:0_18:1, and PC 18:0_18:1, alongside decreases in related DAG and LPC species, as well as select ceramide species (e.g., Cer d18:1_16:0 and Cer d18:1_18:1).

Collectively, these data demonstrate that TgSEC14-LTP1 is important for maintaining PC-centered lipid homeostasis and for preserving lipid pools enriched in unsaturated acyl chains that are normally derived from the host, including C20:4 and C18:1, thereby supporting the lipid balance required for parasite growth and survival. These steady-state changes motivated subsequent binding and metabolic flux experiments to define how TgSEC14-LTP1 influences the availability of DAG- and PC-related precursors to the parasite.

Stable isotope tracing reveals impaired host-to-parasite phospholipid flux with a disproportionate defect in host-derived DAG accumulation in TgSEC14-LTP1-deficient parasites

To assess the function of TgSEC14-LTP1 in host lipid scavenging, we performed a metabolic flux assay based on [U-¹³C₆] glucose labeling of host lipids, as described by Dass et al.⁵⁰ (Figure 4C). Non-confluent host cells were cultured in the presence of [U-¹³C₆] glucose to fuel fatty acid synthesis (FAS I and elon gase pathways) through the generation of the precursor acetyl-CoA, thereby producing ¹³C-pre-labeled host metabolites, including most lipids and fatty acids.⁵² The resulting ¹³C-labeled host cells were subsequently infected with iKOTgSEC14-LTP1 parasites and maintained in a medium free of ¹³C glucose [instead containing unlabeled (¹²C) glucose] to allow specific monitoring of host-scavenged lipids by the parasite. Parasites, either untreated or treated with Rapa, were cultured for 24 h before lipid extraction and analysis by LC-MS to determine the proportion of ¹³C incorporation into individual phospholipid species.

This approach revealed that, in the absence of TgSEC14-LTP1, host-derived ¹³C labeling detected in parasite PC (PC 34:1 and 36:2) and DAG (DAG 34:1, 36:1, and 36:2) was significantly reduced by approximately 20% and 40%, respectively, compared to wild-type parasites or the parental DiCre strain (Figure 4D). Notably, Rapa treatment also reduced labeled PC in the DiCre control, likely due to inhibition of mTORC1 by the rapamycin-FKBP12 complex, which can influence phospholipid synthesis and remodeling.⁵³ Further significant yet milder decreases were detected for PE (40% for PE 34:2), PI (29% for PI 34:1), and PS 36:2 (31%). The pronounced reduction in labeled DAG, together with decreases in PC, PE, PI, and PS, supports the conclusion that TgSEC14-LTP1 is required for efficient accumulation and or retention of host-derived DAG in parasite lipid pools, with downstream consequences for connected glycerophospholipid pathways. Given the central role of DAG in both the Kennedy and CDP-DAG pathways,⁵⁴ reduced host-derived DAG availability provides a parsimonious explanation for coordinated changes in multiple classes, while leaving open the possibility that TgSEC14-LTP1 also influences the availability or remodeling of additional PV-associated lipid pools.

TgSEC14-LTP1 promotes parasite-associated accumulation and LD partitioning of fluorescent PC analog signal

Given the effects of TgSEC14-LTP1 depletion on LD abundance and parasite lipid composition, we next examined the parasite associated accumulation of fluorescent phospholipid analogs. We examined NBD-PC (NBD-PC 18:1–12:0) and NBD-PA (NBD-PA 18:1–12:0), two phospholipids linked to membrane biogenesis and LD formation.⁵⁵ HFFs infected for 18 h with either WT + Rapa or iKDTgSEC14-LTP1 −/+ Rapa parasites were incubated with NBD-labeled lipids for an additional 6 h, followed by fixation and immunofluorescence staining of intracellular parasites. For extracellular (released) parasites, infected cells were lysed after the 6-h NBD-PA/PC pulse-chase, and the released parasites were collected and mounted on coverslips for analysis (Figures 5A–5F).

Figure 5. TgSEC14-LTP1 deletion impairs PC uptake.

(A) Workflow of the uptake phospholipids assay.

(B) Uptake assay showing the incorporation of NBD-labeled phosphatidic acid (NBD-PA) and phosphatidylcholine (NBD-PC) in intracellular and parasites released after the 6-h intracellular pulse-chase. WT + Rapa and iKDTgSEC14-LTP1 −/+ Rapa strains were analyzed. Scale bars: 5 and 2 μm, respectively.

(C–F) Quantification of NBD-PA (C and D) and NBD-PC (E and F) fluorescence in intracellular (C and E) and released (D and F) parasites of WT + Rapa and iKDTgSEC14-LTP1 − /+ Rapa strains, assessing lipid uptake efficiency (n = 4; significance was tested by one-way ANOVA; mean ± SE; (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).

NBD-PA accumulated in membranes and intracellular puncta of both WT and TgSEC14-LTP1-depleted parasites with no obvious qualitative deficit (Figure 5B, NBD-PA). In contrast, NBD-PC signal within parasite intracellular puncta was significantly reduced in TgSEC14-LTP1-depleted parasites, with an 89% decrease in NBD-PC fluorescence compared to control parasites (Figure 5B, NBD-PC; Figure 5E). All images were acquired using identical exposure, laser power, and gain settings. Under these matched conditions, control parasites displayed prominent NBD-PC-positive puncta and reduced residual-host-associated NBD-PC signal near the PV, whereas TgSEC14-LTP1-depleted parasites showed diminished parasite puncta with comparatively higher signal near the PV. Together with the unaltered NBD-PA pattern, these results indicate a selective defect in parasite-associated accumulation of the PC analog rather than a global inability to associate with fluorescent lipids.

Quantification in released parasites supported the intracellular findings, with TgSEC14-LTP1-depleted parasites having significantly reduced NBD-PC fluorescence (Figure 5B, NBD-PC; Figure 5F).

Time course imaging further showed that in control parasites (WT (+) and iKD TgSEC14-LTP1 (−)), NBD-PC first appeared in parasite membranes and subsequently in intracellular puncta that increased in number over 4 to 6 h, whereas TgSEC14-LTP1-depleted parasites displayed markedly reduced NBD-PC-positive puncta formation (Figure S7A). Nile red co-staining indicated that the NBD-derived signal accumulates in LD-associated puncta observed in the parasite, consistent with partitioning of probe-derived material into neutral-lipid storage compartments (Figure S7B).

Importantly, preserved NBD-PA accumulation in Rapa-treated parasites argues against a generalized defect in fluorescent lipid association or gross viability.

Because LPC can be generated from PC, we also assessed parasite-associated accumulation of an NBD-LPC probe (Figures S8A–S8D). Upon TgSEC14-LTP1 depletion, NBD-LPC fluorescence decreased by ~30% relative to the WT + control, whereas the NBD-PC signal was reduced more strongly, by 89% for NBD-PC 18:1_12:0 and 55% for NBD-PC 16:0_12:0 (Figures 5E, S8C, S8E, and S8F). Because acyl-chain composition and fluorophore placement influence membrane partitioning and metabolic handling of NBD phospholipids, differences between NBD-PC species should be interpreted cautiously and do not, on their own, establish differences in TgSEC14-LTP1 binding kinetics. Nevertheless, across the probes tested, TgSEC14-LTP1 depletion had a larger effect on parasite-associated accumulation of PC analog signal than on LPC analog signal, consistent with a disproportionate requirement for TgSEC14-LTP1 in PC-linked lipid handling under these conditions.

Together, these results indicate that TgSEC14-LTP1 is required for efficient parasite-associated accumulation of NBD-PC-derived signal in LD-associated puncta and are consistent with the reduced PC abundance observed by lipidomics and the reduced host-to-parasite labeling of PC and DAG pools detected by stable isotope labeling.

DAG and PC co-purify with TgSEC14-LTP1

To investigate whether there is a direct interaction/association between TgSEC14-LTP1 and lipid molecules, we conducted a lipid-trap mass spectrometry approach using parasites expressing either TgSEC14-LTP1-Myc or GRA1-Myc as a negative control (Figure 6A). GRA1 was chosen as a negative control because, similar to TgSEC14-LTP1, it is derived from dense granules and secreted into the PV; however, unlike SEC14 proteins, it has not been reported to contain a lipid-binding domain. Myc-tagged proteins were immunopurified using Myc-Trap agarose from intracellularly growing parasites, with the aim of capturing the secreted pool of TgSEC14-LTP1-Myc in a native, ligand-associated state. The efficiency of purification was assessed by silver staining, Coomassie blue staining, and immunoblot analysis (Figures 6B–6D). Note the negative silver staining of GRA1-Myc (Figure 6C), likely due to its low cysteine content and a very low isoelectric point (3.9). Lipids co-isolating with the immunopurified proteins were extracted and analyzed with mass-spectrometry-based lipidomics. The results revealed selective enrichment of DAG and PC in TgSEC14-LTP1-Myc pull-downs compared with GRA1-Myc controls (Figures 6E and 6F), despite the slightly higher purification yield for GRA1-Myc (Figures 6B–6D).

Figure 6. TgSEC14-LTP1 co-purifies with DAG and PC.

(A) Workflow for lipid-trap mass spectrometry analysis to identify lipids associated with TgSE C14-LTP1-Myc- or GRA1-Myc-tagged proteins.

(B and C) TgSEC14-LTP1-Myc (A) or GRA1-Myc (B) were purified from the RH DiCre iKOTgSE C14-LTP1-HA, TgSEC14-LTP1-Myc, and RH Δku80 GRA1-Myc strains, respectively. Samples from the eluted fractions (E1–E4) were analyzed by silver staining and immunoblot using an anti-Myc antibody. IN, input; and FT, flowthrough.

(D) Coomassie staining of the eluted fractions corresponding to TgSEC14-LTP1-Myc and GRA1-Myc immunopurification.

(E and F) Lipidomic analyses from immunoprecipitated TgSEC14-LTP1-Myc and GRA1-Myc. Bar graphs show the quantity of the indicated lipids detected with TgSEC14-LTP1-Myc and GRA1-Myc by GC-MS. Data represent the mean ± SD of technical replicates (n = 2). p values corresponding to the two-way ANOVA followed by Sidak’s multiple comparisons test are indicated (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001).

Among the most enriched lipid species in the TgSEC14-LTP1 pull-down were multiple PC species and corresponding DAG derivatives, including PC(36:2)/DAG(18:1_18:1), PC(36:1)/DAG (18:0_18:1), PC(34:2)/DAG(16:0_18:2), PC(34:1)/DAG(16:0_18: 1), PC(38:5)/DAG(18:1_20:4), and PC(36:4)/DAG(16:0_20:4) (Figure 6F). Notably, these same species, or closely related forms, are among the most depleted lipid pools in TgSE C14-LTP1 knockdown parasites, including PC 18:1_18:1/PC 36:2, PC 16:0_18:1, PC 14:0_16:0, PC 36:3, PC 18:0_18:1, PC 38:4, and multiple DAG species such as DAG 18:1_18:1, DAG 16:0_18:1, and DAG 18:1_18:2 (Figures S6A and S6B). Together, these observations indicate that TgSEC14-LTP1 preferentially co-purifies with DAG and PC species enriched for acyl chains commonly observed in host-derived and scavenged lipid pools.

The relative abundance of lipids recovered with TgSEC14-LTP1 does not necessarily report binding affinity, as recovery can be influenced by endogenous lipid abundance, extraction efficiency, and ionization properties. Nevertheless, the selective co-purification of PC and DAG with TgSEC14-LTP1, together with the depletion of these pools in TgSEC14-LTP1-deficient parasites and the reduced host-to-parasite labeling of DAG and PC observed by isotope tracing, supports the conclusion that TgSEC14-LTP1 engages PC and DAG in vivo and promotes the maintenance of PC and DAG pools required for parasite growth.

TgSEC14-LTP1 shares structural similarity with SEC14 proteins and contains a hydrophobic-lipid-binding cavity

To explore how TgSEC14-LTP1 may engage phospholipids, we used AlphaFold3 to predict an apo structure, which displayed good confidence in fold prediction, with pLDDT scores above 70 (Figure S9A). Structural comparison to crystal structures of Sec14 proteins and close Sec14 homologs indicated that TgSEC14-LTP1 adopts a similar overall fold (Figure S9B). Proteins in the SEC14/CRAL-TRIO domain superfamily bind lipophilic molecules through a large hydrophobic pocket embedded within an α/β architecture and a mobile helical gate that regulates ligand access to the pocket.^38,56 The predicted secondary structure of TgSEC14-LTP1 is composed of 10 α-helices (α1–α10) and 5 β-strands (β1–β5) (Figure 7A). Comparative sequence analysis with the CRAL-TRIO domain of Kalirin (PDB ID: 7UR2) and yeast SEC14p (PDB ID: 3B74)^37,38 shows that helices α3, α4, α5, α6, and β-strands β2 and β3 contribute to the predicted cavity wall and gate region (Figures 7A and 7C).

Figure 7. Structural analysis of TgSEC14-LTP1 and its interaction with phosphatidylcholine (PC) and diacylglycerol (DAG).

(A) Schematic representation of the secondary structure of TgSEC14-LTP1. Alpha helices (α) are shown as yellow rectangles, and beta strands (β) are depicted as blue arrows. The conserved lipid-binding domain characteristic of SEC14 proteins is depicted alongside a sequence alignment of representative SEC14-domain-containing proteins. Black arrows indicate amino acid residues involved in PC and DAG binding observed in the docking below.

(B) Molecular docking analysis of AlphaFold TgSEC14-LTP1 prediction with PC or DAG, performed using homology modeling of PC with PDB: 3B7Q used as a reference model for phospholipid binding. Upon placement, Coot was used to remove clashes and locally minimize the binding site.

(C) Surface representation of TgSEC14-LTP1, color-coded by hydrophobicity. Aqua blue indicates less hydrophobic regions, while yellow highlights more hydrophobic areas. The hydrophobic cavity is boxed, with PC docked within this region.

To evaluate whether the predicted pocket is geometrically compatible with DAG and PC, we performed molecular docking using homology modeling and energy minimization of phospholipids on the AlphaFold3 prediction of TgSEC14-LTP1. Docking simulations revealed that TgSEC14-LTP1 can accommodate both DAG or PC and that the residues including M130, M132 in β3, Y83 in β2, F168, and F164 may play an important role in gating the phospholipid as well as selectively binding to the choline moiety (Figure 7B). However, because side-chain placements and gate dynamics are uncertain in predicted structures, we interpret the docking results qualitatively as support for ligand compatibility rather than as evidence for residue-level specificity.

Additionally, phylogenetic analysis and sequence alignments further indicate substantial divergence among SEC14 domain proteins across species (Figures 1A and S1A). While residues shaping the predicted hydrophobic pocket are conserved among TgSEC14-LTP1 homologs (Figures 7A and S1A), divergence between apicomplexans and mammals (Figures 1A and S1A) underscores the potential for selective targeting.

Taken together, these results support the conclusion that TgSEC14-LTP1 folds as a divergent SEC14/CRAL-TRIO-like domain with a conserved hydrophobic pocket compatible with binding of DAG-and PC-sized ligands.

In summary, our data identify TgSEC14-LTP1 as a secreted dense-granule protein essential for intracellular growth. Loss of TgSEC14-LTP1 reduces host-derived lipid flux into parasite pools, with prominent effects on DAG and PC and more modest effects on related species such as LPC and free FA. Lipid copurification with TgSEC14-LTP1, together with structural modeling and docking, supports in vivo association with PC and DAG. Overall, these findings support a model in which TgSEC14-LTP1 functions as a PV lumen SEC14-domain lipid-binding factor that promotes maintenance of PC and DAG pools required for membrane biogenesis and parasite division, highlighting TgSEC14-LTP1 as a promising target for antiparasitic therapy.

DISCUSSION

The growth of Toxoplasma and the expansion of the parasitophorous vacuole (PV) rely on a continuous lipid supply derived from both host resources and parasite biosynthetic pathways.^57–59 While multiple routes for host lipid acquisition have been described, including vesicle-associated delivery of host material to the PV,²¹ the mechanisms that confer selectivity for individual lipid classes and species remain incompletely defined. Because vesicular capture and membrane remodeling can perturb lipid composition and bilayer organization, additional processes are likely to operate at the host parasite interface to regulate lipid availability while preserving membrane integrity and lipid balance. To date, the molecular components that couple host lipid release or remodeling to productive accumulation of defined lipid species within parasite pools remain largely unknown.

Here, we identify TgSEC14-LTP1 as an essential SEC14/CRAL-TRIO-like dense granule protein that is secreted into the PV lumen and required for efficient parasite replication. Our data provide evidence that TgSEC14-LTP1 engages lipid pools relevant to parasite membrane biogenesis. In other eukaryotes, SEC14-domain proteins are lipid-binding modules that coordinate lipid exchange, metabolism, and membrane signaling through ligand-dependent conformational transitions.^60–66 Structural modeling indicates that TgSEC14-LTP1 adopts a SEC14-like fold and contains a hydrophobic pocket compatible with phospholipid-sized ligands, consistent with the conserved architecture of this superfamily.^33,37,61,62 In this context, TgSEC14-LTP1 represents a mechanistically plausible candidate to link lipid binding in the PV lumen to downstream effects on parasite lipid composition.

Multiple lines of functional evidence support a central role for TgSEC14-LTP1 in PC-centered lipid homeostasis. Steady-state lipidomic analyses revealed a selective depletion of PC species and metabolically connected pools, including DAG and LPC species enriched for unsaturated acyl chains that are typically host-derived. Stable isotope tracing using host cells pre-labeled with [¹³C] glucose provided stringent measure of host-to-parasite flux, since parasite labeling can only arise through acquisition of host-synthesized lipids. Under these conditions, TgSEC14-LTP1-deficient parasites exhibited a disproportionate reduction in host-derived DAG label (~40% reduction), with a concurrent reduction in host-derived PC label (~20% reduction), consistent with impaired delivery or maintenance of these host-derived lipid inputs to parasite lipid pools. In parallel, fluorescent lipid pulse experiments showed a selective defect in parasite-associated accumulation of an NBD-PC analog, while NBD-PA accumulation was largely preserved, supporting substrate selectivity at the level of PC-linked lipid handling rather than a general defect in fluorescent lipid association. Finally, lipid-trap mass spectrometry demonstrated selective co-purification of DAG and PC with TgSEC14-LTP1 from intracellular parasites, consistent with in vivo association of TgSEC14-LTP1 with these lipid classes.

These findings support a model in which TgSEC14-LTP1 functions in the PV lumen to promote the availability of DAG- and PC-related pools that feed parasite phospholipid biogenesis. The specific steps influenced by TgSEC14-LTP1 remain to be defined and could include sequestration and retention of host-derived lipids within the PV lumen, presentation of lipid substrates to PV-localized enzymes, or facilitation of lipid remodeling reactions that generate precursors for bulk synthesis. Consistent with related models proposed in Plasmodium falciparum, host membrane PC may be accessed directly and/or converted into downstream intermediates that contribute to parasite phospholipid synthesis.^25–29 One possibility is that host phospholipase C (PC-PLC) activity contributes to conversion of PC to DAG,⁶⁷ thereby enriching DAG in pools accessible to the PV lumen. In this framework, TgSEC14-LTP1 could bind PC or DAG and influence their partitioning into parasite-accessible pools. Once in PV-associated pools, these lipids could undergo additional processing by secreted lipases or phospholipases⁶⁸ or be further remodeled after incorporation into the parasite, generating LPC, FFA, DAG, and head group metabolites that support bulk PC synthesis and membrane expansion.^{14,25,27,50,69}

The lipid perturbations observed upon TgSEC14-LTP1 depletion provide a coherent explanation for the cellular phenotypes. Reduced PC availability would be expected to constrain membrane biogenesis during endodyogeny, contributing to smaller parasites and reduced PV expansion. A pronounced reduction in host-derived DAG flux is also consistent with broader effects on glycerophospholipid biogenesis, given the central role of DAG as a backbone precursor in multiple biosynthetic pathways.⁵⁴ The concomitant reduction in FFAs is likely to limit neutral lipid storage and LD biogenesis, consistent with the marked decrease in LD abundance observed in TgSEC14-LTP1-deficient parasites. In response to PC limitation, the parasite appears to engage compensatory remodeling pathways, including increased PE and LPE and an increase in SM, which may partially substitute for PC-like structural roles in membranes. Shifts in ceramide composition toward longer and more unsaturated forms may reflect altered substrate availability for sphingolipid metabolism or compensatory adjustments linked to SM abundance. These compensatory responses resemble remodeling programs described under PC-limiting conditions in other apicomplexan systems and emphasize the centrality of PC homeostasis for parasite viability.^29,70,71

An important feature of the TgSEC14-LTP1 phenotype is its apparent selectivity. The preservation of NBD-PA accumulation in TgSEC14-LTP1-depleted parasites, together with the preferential reduction in parasite-associated accumulation of NBD-PC, argues that TgSEC14-LTP1 does not broadly control phospholipid handling but instead disproportionately affects PC-linked pools. Lipid-trap mass spectrometry further supports engagement with DAG and PC. At the same time, co-purification reflects association under the extraction conditions and may include contributions from pre-secretion pools in dense granules. Additional experiments that interrogate PV-lumen-enriched TgSEC14-LTP1, or that test cavity mutants predicted to disrupt ligand association, will be useful to strengthen the causal link between lipid engagement and the in vivo flux phenotypes.

Two important caveats remain. First, while isotope tracing and fluorescent lipid assays demonstrate reduced host-derived lipid flux into parasite pools, they do not by themselves establish the precise step at which TgSEC14-LTP1 acts nor the directionality of any lipid exchange event. Bidirectional exchange, sequestration, and remodeling-dependent retention are all plausible. Second, the observed enrichment of DAG in lipid pulldown assays may reflect the formation of stable post-hydrolysis complexes during detergent-based protein extraction, rather than an intrinsic binding preference for DAG.

Identification of interacting partners, including candidate phospholipases or other lipid-modifying enzymes, may clarify whether TgSEC14-LTP1 operates within a multi-protein lipid handling pathway in the PV lumen. Approaches such as live-cell lipid-specific biosensors, kinetic labeling strategies, and targeted mutagenesis of the predicted hydrophobic pocket and gate residues could help define mechanism and directionality.

From a therapeutic perspective, SEC14 pockets are druggable in fungi and plants, and small molecules that disrupt ligand occupancy can block lipid-dependent functions. TgSEC14-LTP1 is essential, accessible within the PV, and divergent from mammalian SEC14 proteins, making it an attractive target for selective intervention. Moreover, SEC14-domain proteins are conserved across apicomplexans, raising the possibility that targeting this family could yield broad-spectrum antiparasitic strategies.

Finally, while TgSEC14-LTP1 has a dominant impact on PC-centered homeostasis, Toxoplasma must acquire and remodel additional lipid classes. We anticipate that other PV-associated lipid-binding proteins share this workload. TgSEC14-LTP2 is a strong candidate for complementary functions and may preferentially influence non-PC lipid pools. Defining TgSEC14-LTP2 substrate specificity and its relationship to TgSEC14-LTP1 will be an important direction for future work.

Limitations of the study

SEC14 proteins are well-known lipid transfer proteins that mediate the trafficking of phospholipids between membranes. Although our data support a role for TgSEC14-LTP1 in regulating lipid flux in T. gondii, direct biochemical demonstration of lipid transfer activity will require additional in vitro assays. Furthermore, characterization of lipid binding specificity using lipid-protein overlay assays or related biochemical approaches would strengthen the mechanistic understanding of TgSEC14-LTP1 function.

RESOURCE AVAILABILITY

Lead contact

Requests for further information and resources should be directed to and will be fulfilled by the lead contact, Jeroen P.J. Saeij (jsaeij@ucdavis.edu).

Materials availability

All materials generated in this study are available upon request.

Data and code availability

• No original dataset is associated with this study.

• The paper does not report original code.

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

STAR★METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Parasite and host cell culture

The study utilized Human Foreskin Fibroblasts (HFF) as the primary eukaryotic cell line for culturing Toxoplasma gondii. HFF cells were originally obtained from Dr. John Boothroyd and originated from a pool of anonymous donors. Cells were authenticated by morphological examination and growth characteristics consistent with ATCC standards and were confirmed to be mycoplasma-free throughout the study. HFF cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing glucose (4.5 g/L), supplemented with 2 mM glutamine, 100 U/mL penicillin,100 μg/mL streptomycin, 10 mM gentamicin, and 10% fetal bovine serum. Cells were maintained in a humidified incubator (37°C, 5% CO2), grown to confluence, regularly harvested by trypsinization, and seeded into dishes, plates, or flasks as needed.

The DiCreRHΔku80 strain was provided by Dr. Sebastian Lourido (Biology Department, Massachusetts Institute of Technology, Cambridge, MA, USA). The parasites were maintained by infecting confluent host cell monolayers and passaged on alternate days. For phenotypic assays, parasitized host cells were scraped 40–48 h post-infection and passed through 27-gauge syringes to release intracellular tachyzoites. Parasites were then counted and used to infect cells at the specified MOI, depending on the assay requirement.

METHOD DETAILS

Molecular cloning

The TgSEC14-LTP1-DiCre-HA-FLAG-HXGPRT strain was generated by transfecting the DiCreRHΔku80 strain with a CRISPR construct expressing Cas9 and gene-specific sgRNA, along with a homology donor amplicon. The amplicon contained the HA-FLAG-LoxP and HXGPRT selection cassette flanked by 40 bp 5^′ and 3^′ homology arms designed for recombination at the TGGT1_215160 locus. It was amplified from the pG152 plasmid template. The TgSEC14-LTP1–3^′UTR-specific sgRNA sequence was cloned by replacing sgUPRT in the pU6-universal vector. Tachyzoites (~2 × 10⁷) were electroporated with DNA (CRISPR plasmid: 25 μg, Amplicon: 8 μg) in filter-sterile Cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2 supplemented with 5 mM glutathione and 5 mM ATP; pH 7.6) using a BIO-RAD GenePulser Xcell. Transgenic parasites were selected with 25 μg/mL mycophenolic acid (Millipore 89287) and 25 μg/mL xanthine (Millipore X3627). Surviving tachyzoites were isolated by limiting dilution, and individual clones were screened by genomic PCR for 5^′ and 3^′ crossover events at the gene locus (primers in Table S1). Clones were further validated for TgSEC14-LTP1-DiCre-HA-FLAG expression by immunostaining.

The bicistronic vector expressing the Cas9 genome editing enzyme and specific sgRNAs targeting the TgSEC14-LTP1 coding sequence was constructed as described previously.⁷³ Briefly, oligonucleotides TgSEC14-LTP1-CRISPR-FWD and TgSEC14-LTP1-CRISPR-REV (Table S1) were annealed and ligated into the pTOXO_Cas9-CRISPR plasmid to create vectors used for construction of T. gondii conditional TGGT1_215160 knockout strain (iKOTgSEC14-LTP1).

To generate the TgSEC14-LTP1 conditional knockout (cKO) plasmid, pUC57Simple-LoxP-Tg215160-LoxP-HXGPRT, the TgSEC14-LTP1^′UTR with a loxP site inserted 100 bp upstream of the TgSEC14-LTP1 start codon, and a recodonized TgSEC14-LTP1 cDNA-HA sequence, were DNA-synthesized by GenScript (GenBank: PV231209) using plasmid pUC57Simple-LoxP-HXGPRT (GenBank: PQ154619) as vector backbone.

To generate the conditional TgSEC14-LTP1 knockout strain (iKOTgSEC14-LTP1), the plasmid pUC57Simple-LoxP-Tg215160-LoxP-HXGPRT was linearized using NotI and cotransfected with pTOXO_Cas9-CRISPRsgTgSEC14-LTP1 into the RH DiCre strain.⁷⁴ Recombinant parasites were selected 24 h post-transfection by addition of 25 mg/mL mycophenolic acid and 50 mg/mL xanthine to culture medium. Resistant parasites were cloned, and successful integration at the TgSEC14-LTP1 locus and absence of the endogenous sequence were confirmed using primer pairs a/b and a/c. Rapa-induced excision of the floxed TgSEC14-LTP1 sequence was confirmed using primer pair a/d.

To generate the iKOTgSEC14-LTP1, P_GRA1⁻TgSEC14-LTP1-Myc complemented strain, the promoter sequence of GRA1 was amplified using primers LICF-Pgra1_F2 and 215160-P_GRA1_R, with RH strain genomic DNA as the template. The coding sequence of TgSEC14-LTP1 was PCR-amplified using primers P_GRA1-215160_F and LICR-TGGT1_215160_R. The resulting PCR fragments were assembled via overlap extension PCR and cloned into the pLIC-Myc-DHFR vector using ligation-independent cloning (LIC), as previously described⁷⁵ yielding the pLIC-P_GRA1-TgSEC14-LTP1-Myc-DHFR vector. RH DiCre iKOTgSEC14-LTP1 parasites were electroporated with the pLIC-P_GRA1-TgSEC14-LTP1-Myc-DHFR vector, and stable transformants were selected using pyrimethamine. Clonal parasite lines expressing the TgSEC14-LTP1-Myc transgene were isolated and treated with Rapa to induce excision of the endogenous TgSEC14-LTP1-HA allele. Parasites expressing TgSEC14-LTP1-Myc, but lacking the TgSEC14-LTP1-HA allele, were selected and cloned by limiting dilution, generating the RH DiCre iKOTgSEC14-LTP1-HA, TgSEC14-LTP1-Myc strain.

To generate the RH Δku80 GRA1-Myc strain, the coding sequence of GRA1 was amplified using primers LICF-GRA1_F and LICR-GRA1_R and RH Δku80 genomic DNA as the template. The PCR product was cloned to pLIC-Myc-DHFR vector, using the LIC cloning method as described in.⁷⁵ The resulting pLIC-GRA1-Myc-DHFR vector was linearized with the BsiWI restriction enzyme and electroporated into the RHΔku80 strain. Knockin parasites were selected using pyrimethamine and cloned by limiting dilution.

Indirect immunofluorescence assays

Confluent HFFs grown on coverslips were infected at an MOI of 2, depending on experimental requirements. Parasitized cells were fixed with 2% paraformaldehyde for 20 min at room temperature and washed three times with PBS. Cells were blocked and permeabilized with blocking buffer containing 3% (wt/vol) BSA, 5% (vol/vol) goat serum, and 0.1% Triton-100 in PBS at room temperature for 20 min. They were then incubated overnight at 4°C with primary antibodies (α-HA, 1:500; α-TgIMC1, 1:2000; α-GAP45, 1:5000; α-TgSag1, 1:5000, α-TgGRA23 1:500) followed by three PBS washes. For secondary staining, cells were incubated with goat anti-rat Alexa Fluor 488, goat anti-mouse Alexa Fluor 594, or goat anti-rabbit Alexa Fluor 594. DNA was stained with 4^′,6-diamidino-2-phenylindole (DAPI) in blocking buffer for one hour at room temperature, followed by three PBS washes. Coverslips were mounted using Mowiol 4–88 buffer (Millipore 81381). Images were acquired using an epifluorescence microscope (Nikon ECLIPSE Ti-S, Japan or ZEISS ApoTome.2).

Lytic cycle assays

Plaque assays

Plaque assays were performed using confluent HFF monolayers seeded in 12-well plates. Cells were infected with 100 parasites per well and incubated for seven days in the presence or absence of Rapa, without perturbation. After incubation, cells were fixed with ice-cold methanol (10 min), stained with crystal violet (15 min), and washed three times with PBS.

Replication and endodyogeny assays

To assess cell division, confluent HFFs grown on coverslips were infected at an MOI of 1 for 24 or 48 h, fixed with paraformaldehyde, and stained with specific antibodies: α-HA and α-TgSAG1 for replication and α-TgIMC1 for endodyogeny. Parasitophorous vacuoles were analyzed for the number of tachyzoites to assess replication. Endodyogeny was assessed by quantifying the fraction of tachyzoites containing IMC1-stained daughter cells.

Invasion assays

To measure invasion efficiency, HFFs were infected at an MOI of 10 for 60 min, fixed with 2% paraformaldehyde (20 min) and washed three times with PBS. Cells were blocked with 2% FBS in PBS, then incubated with α-TgSAG1 antibody. Following three PBS washes, cells were permeabilized with 0.2% Triton X-100 + 2% of FBS in PBS (20 min) and stained with α-TgIMC1, secondary antibodies (Alexa Fluor 488/594), and DAPI. Coverslips were mounted in Mowiol 4–88 buffer. Extracellular and intracellular parasites were distinguished by differential staining: parasites stained with α-TgIMC1 but not α-TgSag1 were classified as intracellular parasites.

Egress assays

For the egress assay, HFFs were infected at an MOI of 1 and cultured for 42–46 h. Cells were then incubated for 3 min at 37°C with A23187 1 μM to induce egress. Egress was quantified by counting tachyzoites stained solely with α-TgSAG1.

TgSEC14-LTP1 and GRA2 colocalization

Confluent HFF cells grown on coverslips were infected at an MOI of 1 with tachyzoites from WT and iKDTgSEC14-LTP1 strains, in the presence or absence of Rap, for 24 h (intracellular state) and 48 h (extracellular state). Intracellular parasites were fixed with 2% paraformaldehyde for 20 min. For extracellular (released) parasites, after 48 h, infected cells were scraped, and intracellular tachyzoites were released by mechanical disruption. The parasites were then centrifuged for 7 min at 570 g, resuspended in PBS, and placed on coverslips pre-coated with 0.1% BSA. Parasites were incubated for 30 min at 37^◦C and subsequently fixed with 2% PFA for 20 min.

For colocalization analysis of GRA2 and iKDTgSEC14-LTP1-HA, coverslips were blocked with blocking buffer at room temperature for 20 min. Subsequently, the cells were incubated overnight at 4^◦C with primary antibodies: rat anti-HA and mouse anti-GRA2, the coverslips were then incubated with Alexa Fluor 488 and Alexa Fluor 594-conjugated secondary antibodies in blocking buffer for 1 h at room temperature. DAPI was used for DNA staining Finally, the cells were mounted and analyzed by fluorescence microscopy.

Parasite lipid droplets staining

Intracellular LDs staining

Parasites precultured with or without Rapa for 48 h were used to infect HFFs with tachyzoites of the WT or iKDTgSEC14-LTP1 strains −/+ Rapa at an MOI of 1 for 24 h, in the absence or presence of 0.3 mM OA-albumin in DMEM +1% FBS. After infection, cultures were fixed with 2% PFA for 20 min and washed three times with PBS.

Extracellular LDs staining

Cells cultured in 6-well plates were infected at an MOI of 3 with WT or iKDTgSEC14-LTP1 -Rapa, and 6 with iKDTgSEC14-LTP1 +Rapa and in the absence or presence of 0.3 mM OA in DMEM +1% FBS. After 24 h of infection, cells were scraped, and parasites were released by passing them once through a 27gauge (G) needle, followed by centrifugation at 570g for 7 min. The parasites were then resuspended in 1 mL of PBS and mounted on coverslips pre-coated with 0.1% BSA. After a 30-min incubation, parasites were fixed with 2% PFA.

All the samples were processed for IFA. LD staining was performed after IFA processing. Coverslips were incubated with 250 μL of 30 μM BODIPY 493/503 or Nile red for 30 min at 37°C, followed by three washes with PBS. Finally, coverslips were mounted and analyzed by fluorescence microscopy.

Uptake assays of neutral lipids

HFF cells pretreated with 0.15 mM OA for 24 h were incubated with BODIPY 493/503 for 1 h, followed by thorough washing with PBS. The cells were then incubated for 3 h in fresh DMEM (without additional OA or BODIPY) to allow further processing or redistribution of the labeled lipids. Subsequently, cells were infected with the WT + Rapa or iKDTgSEC14-LTP1 −/+ Rapa parasites for 24 h before fixation for immunostaining. Lipid uptake was quantified by calculating the ratio of LDs number per parasite.

Lipidomic analysis of iKDTgSEC14-LTP1 strain

Expression analysis of TgSEC14-LTP1 revealed that after 48 h of treatment with Rapa and subsequent reinfection of HFFs for 24, the protein expression was completely abolished. In a triplicate experiment, parasites were grown for 24 hours in presence or absence of Rapa within a confluent HFF monolayer in 75 cm2 flasks. Intracellular tachyzoites were harvested and metabolically quenched by rapid chilling in a dry ice-ethanol slurry bath, followed by centrifugation at 4°C. After metabolic quenching, HFFs were lysed by passing them through 25 and 27 G syringes and subsequently filtered using a 5 μm pore-size membrane. The parasite pellet was washed three times with ice-cold PBS, and aliquots containing 1 × 10⁷ parasites were stored for further analysis. Toxoplasma lipids were extracted using acetonitrile/isopropanol/water mixture (3:3:2 v/v/v) on ice, followed by separation. For lipid profiling, metabolite extraction was performed on ice with standards added in the presence of methanol (MeOH) and methyl tert-butyl ether (MTBE). Subsequently, the samples were dried and resuspended in 108.6 μL MeOH: toluene (90:10, (v/v) containing CUDA (12-[[(cyclohexylamino)carbonyl] amino]-dodecanoic acid, 50 ng/mL). High-resolution mass spectrometric analysis was performed using an Agilent 6534c quadrupole time-of-flight mass spectrometer equipped with a dual electrospray ionization (ESI) source. Simultaneous MS1 and data dependent MS/MS acquisition were used. Post-acquisition data processing was carried out using MS-DIAL software (version 2.69). Lipid quantification was achieved by integrating the two most abundant ion adducts for each lipid species, followed by normalization to the sum of all internal standards.

Lipidomic analysis of iKOTgSEC14-LTP1 strain

Parasites were cultured on HFF monolayers in 175 cm² flasks under standard conditions and either left untreated or treated with 50 nM Rapa for 24 or 48 h. Each condition was performed in quadruplicate, with one flask per replicate. Infected HFF cells were harvested by scraping and immediately metabolically quenched in a dry ice-ethanol slurry bath. Parasites were mechanically released using a 26-gauge syringe needle and passed through a 3 μm filter to remove host cell debris. The parasites were then washed three times with ice-cold PBS by centrifugation at 900 × g for 10 min at 4°C, yielding approximately 10⁷ to 10⁸ tachyzoites per replicate. The resulting parasite pellet was snap-frozen in liquid nitrogen and stored at −80°C until lipidomic analysis. Total lipids were extracted using a chloroform/methanol/water mixture (1:3:1, v/v/v) containing PC (C13:0/C13:0) (10 nmol) and C21:0 (10 nmol) as internal standards. Extraction was performed at 4°C for 1 h with periodic sonication. Polar and apolar metabolites were separated by phase partitioning via the addition of chloroform and water to achieve a final solvent ratio of chloroform/methanol/water (2:1:0.8, v/v/v). For lipid analysis, the organic phase was dried under a stream of nitrogen gas and resuspended in 1-butanol at a concentration of 1 μL butanol per 10⁷ parasites.

Lipids were analyzed as previously described.⁷⁶ Briefly, dried down samples were reconstituted in 80 μL methanol and incubated at 30°C for 5 min with vigorous vortex. Samples (1 μL) were analyzed by LCMS (Agilent 1290 infinity/Infinity II Agilent) MS (Agilent 6495c triple quadrupole) in our platform GEMELI. Acquisition DMRM (dynamic multiple reaction monitoring) method used was referred to as described previously⁷⁷ with a modification for Toxoplasma lipid species. The resulting LCMS data was subjected to targeted analysis using Mass Hunter Quantification software (Agilent). Each lipid species was quantified using a calibration curve of each representative lipid with known abundance. Then each lipid abundance was normalized according to the cell ratio.

iKOTgSEC14-LTP1 lipid profile analysis

For phospholipid analysis, total lipids extracted were separated by one-dimensional silica gel high-performance thin-layer chromatography (HPTLC, Merck) in the presence of 1 nmol phosphatidic acid (PA) (C17:0/C17:0) (Avanti Polar Lipids). To analyze total phospholipids (PL), diacylglycerol (DAG), triacylglycerols (TAG), free fatty acids (FFA), and cholesteryl esters (CE), lipid fractions were resolved by 1D-HPTLC using a hexane/diethyl ether/formic acid (80:20:2, v/v/v) solvent system. Following chromatographic separation, individual lipid spots were scraped from the HPTLC plate, and lipids were methanolized in 200 μL of 0.5 M methanolic HCl at 85^◦C for 3 h in the presence of 1 nmol pentadecanoic acid (C15:0) as an internal standard. The resulting fatty acid methyl esters (FAMEs) were extracted with hexane and subsequently analyzed by gas chromatography-mass spectrometry (GC-MS) (Agilent).

NBD-PA, NBD-PC, and LysoNBD-PC staining

For intracellular parasites, host cells were infected with WT + Rapa and iKDTgSEC14-LTP −/+ Rapa parasites for 18 h. Following infection, the cells were incubated in DMEM without FBS or in DMEM supplemented with 1% delipidated serum containing NBD-PA 5 μM, LysoNBD-PC or NBD-PC 5 μM and grown for an additional 6 h. Cells were then fixed with 2% PFA in 1xPBS and subsequently processed for IFA. For extracellular parasites, cells cultured in 12-well plates were infected at an MOI of 3 with WT + Rapa or iKDTgSEC14-LTP1-Rapa parasites, and MOI of 6 with iKDTgSEC14-LTP1+Rapa. After 24 h of infection, the culture medium was replaced with DMEM without FBS or DMEM with 1% delipidated serum containing 5 μM NBD-PA or 5 μM NBD-PC, and cells were grown for an additional 6 h. Next, cells were scraped and parasites were released by passing them once through a 27G needle. The parasites were then centrifuged at 570xg for 7 min, resuspended in 1 mL of PBS, and mounted on coverslips pre-coated with 0.1% BSA. After a 30 min incubation, parasites were fixed with 2% PFA. Coverslips were then blocked with blocking buffer and processed for IFA.

Host-derived lipids stable isotope labeling

To assess the role of TgSEC14-LTP1 in parasite scavenging of host-derived lipids, HFF cells (approximately 1 × 10⁶ cells per replicate) were grown to confluence in glucose-free DMEM supplemented with 800 μM [U-¹³C₆] glucose. The resulting ¹³C-prelabeled HFF monolayers were then infected with iKO-TgSEC14-LTP1 parasites, either untreated or treated with 50 nM rapamycin, in DMEM containing unlabeled (¹²C) glucose with or without rapamycin. After 24 h, intracellular parasites were metabolically quenched, and their lipid content was analyzed by LCMS as described above. The extent of ¹³C incorporation into phospholipids (% carbon incorporation) was determined from the mass isotopomer distribution (MID) of each FAME using Mass Hunter software (Agilent).

Lipid-trap mass spectrometry

To study protein-lipid interactions, approximately 6 × 10⁹ intracellular tachyzoites of RH DiCre iKOTgSEC14-LTP1-HA, TgSEC14-LTP1-Myc, or RH Δku80 GRA1-Myc strains were harvested by centrifugation at 900 × g for 10 min at 4^◦C and subsequently washed in ice-cold PBS. The resulting cell pellet (~2.5 mL) was resuspended in 5 mL of lysis buffer (20 mM Tris-HCl, pH 8.0, 500 mM KCl, 20% glycerol, 0.2 mM EDTA, 1.5 mM MgCl₂, 0.05% NP-40, and protease inhibitors) and incubated on ice for 10 min. Lysis was performed by Dounce homogenization using a tight plunger (B) with 10 gentle strokes. Lysates were stored at −80°C until further processing. For chromatin digestion, 5 μL of DNase I (1 U/μL, Fermentas, #EN0521) was added, and the sample was incubated at 4°C for 1 h. The lysate was clarified by centrifugation at 20,000 × g for 1 h at 4°C. The resulting supernatant was collected and incubated with 500 μL of Myc-Trap Agarose beads at 4°C for 1 h with gentle rotation (5 rpm). Beads were washed with 20 volumes (~15 mL) of lysis buffer, and bound proteins were eluted in three sequential fractions (E1 to E4) using 500 μL of 2× Myc peptide (100 μg/mL) in elution buffer (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.05% NP-40, 20% glycerol, and 1 mM EDTA).

Lipidomic analyses were performed as described above in the Gemeli Platform.

Sequence alignment and phylogenetic tree

To construct the phylogenetic tree, the tertiary structure of the TgSEC14-LTP1 (TGGT1_215160) protein (AF-S7UQV7-F1-v4) was retrieved from the AlphaFold database or modeled on the google AlphaFold3 server.^73,78 This structure was aligned with homologous protein structures obtained from the Foldseek database,⁴³ which utilizes a discretization approach to encode query structures into sequences based on the 3Di alphabet. High-scoring hits from the Foldseek analysis, along with SEC14 protein sequences from Rattus norvegicus, Neospora caninum, Cystoisospora suis, Sarcocystis calchasi, Besnoitia besnoiti, and Homo sapiens, were subjected to multiple sequence alignment and phylogenetic reconstruction. The Maximum Likelihood phylogenetic tree was built using the Neighbor Joining construction method in CLC Genomics Workbench (version 24.0.1) CLC Genomics Workbench was used to align the sequences with the following IDs: GenBank: RZB79478.1 Glycine soja; GenBank: KRH29416.1 Glycine max; GenBank: AAP51934.1 Oryza sativa Japonica; GenBank: AED96728.1 Arabidopsis thaliana; GenBank: ONM53302.1 Zea mays; GenBank: CAA33511.1 Saccharomyces cerevisiae; GenBank: KXX77290.1 Madurella mycetomatis; GenBank: OCT54775.1 Cladophialophora carrionii; GenBank: ERS97512.1 Sporothrix schenckii; GenBank: AAO21869.1 Homo sapiens; GenBank: 7UR2 Rattus norvegicus; GenBank: EPR60090.1 Toxoplasma gondii; GenBank: CAA93167.1 Schizosaccharomyces pombe; GenBank: XP_003884817.1 Neospora caninum; GenBank: XP_067925371.1 Cystoisospora suis; GenBank: CAL7862735.1 Sarcocystis calchasi; and GenBank: XP_029221031.1 Besnoitia besnoiti.

Tertiary structure analysis

The AlphaFold-predicted tertiary structure model was further analyzed for structural comparison with the Kalirin protein (PDB: 7UR2) and S. cerevisiae (PDB: 3B74) to identify the hydrophobic cavity and potential lipid-binding sites of TgSEC14-LTP1. Structural comparisons and visualization, including the 3D reconstruction of the protein’s hydrophobic surface, were performed using ChimeraX (version 1.8). The PC-based binding structure of TgSEC14-LTP1 was modeled using its AlphaFold-predicted tertiary structure (AF-S7UQV7-F1-v4) and the chemical structure of PC (PDB: 6PL) as the ligand. Docking simulations were performed with Coot.⁷²

QUANTIFICATION AND STATISTICAL ANALYSIS

Data shown in the graphs are presented as the means with standard deviation of at least three experiments where wild-type and mutant strains were used and compared under the experimental conditions mentioned. Statistical analyses were performed using GraphPad Prism 10 software. Significance was tested by one-way Anova test or by unpaired t test, as specified (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). Statistical analysis was done using Welch’s t test and multiple unpaired t-tests.

Supplementary Material

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2026.117299.

KEY RESOURCES TABLE.

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
anti-TgGRA23	Dr. Tatsunori Masatani (Lab. of Zoonotic Diseases, Faculty of Applied Biological Sciences, Gifu University)	N/A
anti-TgSAG1	Dr. John Boothroyd (Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA)	N/A
anti-TgIMC1	Dr. Gary Ward (Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT, USA)	N/A
anti-HA	Invitrogen Thermo Fisher Scientific	CAT# 11867423001;RRID: AB_390918
Alexa Fluor 594 goat anti-mouse	Thermo Fisher Scientific	CAT# A11005; RRID: AB_2534073
Alexa Fluor 594 goat anti-rabbit	Thermo Fisher Scientific	CAT# A11037; RRID: AB_2534095
Alexa Fluor 488 goat anti-rat	Thermo Fisher Scientific	CAT# A21208; RRID: AB_2535794
anti-GAP45	Dr. Con J M Beckers (Department of Cell and Developmental Biology, 108 Taylor Hall, University of North Carolina, Chapel Hill, NC 27599, USA.)	N/A
Anti-GRA1	Dr. Jean-François Dubremetz (Institut National de la Santé et de la Recherche Médicale, Unité 42, Villeneuve d’Ascq, France)	N/A
Chemicals, peptides, and recombinant proteins
BODIPY 493/503	Thermo Fisher Scientific	CAT# D3922
Nile red	Thermo Fisher Scientific	CAT# N1142
NBD-PA (18:1-12:0)	Avanti Polar Lipids	CAT# 810176P
NBD-PC (18:1-12:0)	Avanti Polar Lipids	CAT# 810133P
NBD-PC (16:0_12:0)	Avanti Polar Lipids	CAT# 810131P
LysoNBD-PC (12:0)	Avanti Polar Lipids	CAT# 810128
Oleic Acid-Albumin	Millipore Sigma	CAT# O3008
Experimental models: Cell lines
Human foreskin fibroblast (HFF)	Dr. John Boothroyd (Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA)	N/A
Experimental models: Organisms/strains
DiCreRHΔku80	Pieperhoff. et al.44; https://pmc.ncbi.nlm.nih.gov/articles/PMC4474610/	N/A
iKOTgSEC14-LTP1	This paper	N/A
iKDTgSEC14-LTP1	This paper	N/A
PGRA1-TgSEC14-LTP1-Myc	This paper	N/A
RH Δku80 GRA1-Myc	This paper	N/A
Oligonucleotides
Primers used	This paper	Table S1
Recombinant DNA
GRA16-Myc	This paper	N/A
Software and algorithms
Coot	Casañal et al.72; https://pubmed.ncbi.nlm.nih.gov/31730249/	N/A
Foldseek	van Kempen et al.43; https://pubmed.ncbi.nlm.nih.gov/37156916/	N/A
AlphaFold	Varadi et al.73; https://pubmed.ncbi.nlm.nih.gov/34791371/	N/A
BioRender	BioRender	https://www.biorender.com/
GraphPad Prism (v10.0)	GraphPad Software	https://www.graphpad.com/
ImageJ	National Institutes of Health	https://imagej.net/ij/

Highlights.

• TgSEC14-LTP1 is a SEC14/CRAL-TRIO-like protein with a hydrophobic-lipid-binding pocket

• TgSEC14-LTP1 binds PC and DAG and mediates host-to-parasite lipid flux

• Loss of TgSEC14-LTP1 disrupts parasite PC and DAG homeostasis and impairs bulk PC synthesis

• TgSEC14-LTP1 loss impairs lipid droplet formation and parasite biogenesis, limiting growth