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. 2026 Feb 16;125(1):25. doi: 10.1007/s00436-026-08646-w

Anti-Toxoplasma activity of NBMPR is mediated through the inhibition of nucleoside and nucleobase transporters

Ming Pan 1,2,3,✉, Shu-Qin Tang 1, Ceng-Ceng Ge 1, Shi-Jie Fan 1, He-Xin He 1, Man-Man Bian 1, Bang Shen 4, Si-Yang Huang 1,2,3,✉
PMCID: PMC12909333  PMID: 41692887

Abstract

Toxoplasma gondii stands among nature’s most successful eukaryotic pathogens, a status achieved through refined mechanisms for intracellular survival that enable pan-vertebrate host tropism. This ecological dominance derives principally from evolutionary refinements in metabolic plasticity, a modular integration of de novo biosynthesis with high-efficiency nutrient scavenging systems. The pyrimidine biosynthesis pathway is indispensable for robust parasite proliferation. Our previous work demonstrated that exogenous uracil supplementation bypasses the DHO deficiency-induced growth restriction. Here, we reconfirm the functional pyrimidine rescue using an ATC-knockout parasite line, identifying a protective salvage route that maintains pyrimidine pool homeostasis. Pharmacological suppression of T. gondii growth with NBMPR, a potent hENT1 antagonist, confirms that host hENT1 serves as a critical conduit for pyrimidine and purine acquisition by the parasite. Evolutionary analysis identifies TgENT1, TgENT3, TGGT1_359630, and TgAT1 as hENT1 homologs, with TgENT1 demonstrating the strongest phylogenetic clustering with hENT1. The computational models predict interactions between TgENT1 and a spectrum of ligands (NBMPR, nucleosides, and nucleobases), supporting its classification as a broad-specificity nucleoside/nucleobase transporter, consistent with the function of hENT1. The genetic intractability of TgENT1, however, indicates that it likely plays an essential role in parasite proliferation. Our study reveals that NBMPR-mediated inhibition of both TgENT1 and hENT1 disrupts a hierarchical transport cascade essential for T. gondii to acquire pyrimidines and purines from the host, highlighting the promise of a strategy that simultaneously targets both parasite and host nutrient acquisition pathways for improved anti-toxoplasmosis therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00436-026-08646-w.

Keywords: Toxoplasma gondii, Nucleoside and nucleobase transporter, NBMPR, hENT1, TgENT1

Background

As a member of the evolutionarily conserved apicomplexan parasites, Toxoplasma gondii, is an obligate intracellular protozoan that proliferates rapidly as tachyzoites within virtually all nucleated cells of warm-blooded vertebrates (Pan et al. 2017). The extensive host range encompasses domesticated animals, wildlife (Behnke et al. 2016; Jenkins et al. 2025), and approximately one-third of the global human population (Montoya and Liesenfeld 2004). In immunocompetent hosts, acute infection with T. gondii tachyzoites is typically subclinical, though may progress to ocular toxoplasmosis, before establishing latent infection characterized by semi-dormant bradyzoites (Shwab et al. 2018; Dunay et al. 2018). Immunocompromised hosts, however, face potentially lethal consequences due to bradyzoite reactivation and subsequent tachyzoite reconversion, leading to disseminated toxoplasmosis that presents both a critical global health burden and significant agricultural economic losses (Pan et al. 2022).

To successfully colonize diverse host environments, T. gondii has evolved intricate mechanisms of intracellular parasitism (Farhat and Hakimi 2022). A critical adaptation is its sophisticated metabolic networks that coordinate between metabolite synthesis and host-derived nutrient acquisition (Walsh et al. 2022; Rimple et al. 2025). On one hand, the majority of core metabolic pathways remain intact in T. gondii, including glycolysis (Shukla et al. 2018), the tricarboxylic acid (TCA) cycle (Lyu et al. 2023; Silva et al. 2023), gluconeogenesis (Blume et al. 2015). On the other hand, T. gondii lacks certain enzymes in biosynthesis pathways, rendering it an auxotrophic organism that depends on hijacking host resources, such as arginine and aromatic amino acids (Rajendran et al. 2017; Olson et al. 2020; Parker et al. 2019; Fairweather et al. 2021).

De novo nucleotide biosynthesis and salvage pathways are fundamental for cellular survival (Martorelli Di Genova 2024). T. gondii lacks essential enzymes for de novo purine synthesis and must instead salvage purines through two major pathways mediated by hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) (Fig. 1). Between these routes, AK exhibits approximately 10-fold higher enzymatic activity than HXGPRT, suggesting that adenosine salvage plays a more critical role than hypoxanthine and other purine nucleobases (De Koning et al. 2003). Beyond the early-characterized high-affinity hypoxanthine transporter TgNBT1 (De Koning et al. 2003). T. gondii possesses multiple transporters for purine salvage. These include the dual-specificity transporter TgAT1, which employs distinct binding modes for nucleosides and nucleobases (Chiang et al. 1999; Campagnaro et al. 2022), and TgENT1, whose transcriptomic profile suggests a pivotal role in global purine transport and homeostasis (Messina et al. 2025).

Fig. 1.

Fig. 1

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Illustration of pyrimidine biosynthesis and purine salvage pathways in T. gondii. CPSII, carbamoyl phosphate synthetase II; ATC, aspartate transcarbamoylase; DHO, dihydroorotase; DHODH, dihydroorotate dehydrogenase; OPRT, orotate phosphoribosyltransferase; OMPDC, orotidine-5’-monophosphate decarboxylase; UMP, uridine-5’-monophosphate; UPRT, uracil phosphoribosyltransferase; UP, uridine phosphorylase; CTD, cytidine deaminase; HXGPRT, hypoxanthine-xanthine-guanine phosphoribosyltransferase; AK, adenosine kinase

For pyrimidine biosynthesis, T. gondii relies indispensably on the de novo pathway, as evidenced by the severe uracil auxotrophy resulting from the disruption of any key enzyme, including carbamoyl phosphate synthetase II (CPSII), dihydroorotase (DHO), orotate phosphoribosyltransferase (OPRT), or orotidine-5’-monophosphate decarboxylase (OMPDC) (Fig. 1). Nevertheless, this auxotrophy can be functionally rescued by uracil supplementation (Fox and Bzik 2002, 2015; Fox et al. 2011; Pan et al. 2024), demonstrating the existence of a functional pyrimidine nucleobase transport mechanism in the parasite. The single transporter TgUUT1 exhibits high affinity for both uridine and uracil, and this pyrimidine salvage pathway is a recognized target for new anti-T. gondii treatments (Elati et al. 2023).

To investigate nucleoside/nucleobase transport in T. gondii, we focused on the pyrimidine pathway due to its unique amenability for dissecting both de novo synthesis and salvage mechanisms. Using an integrated approach, including pharmacology, phylogenetics, molecular docking, and genetics, we systematically identified and characterized key transporters. Our work expands the known repertoire of the parasite’s transport systems and establishes a therapeutic paradigm of concurrently halting de novo and salvage nucleotide biosynthesis to combat toxoplasmosis.

Materials and methods

Parasites and HFF cell line

T. gondii type I strain RH-TIR1, TATi, RH, and type II strain Pru, were utilized for transgenic construction. All T. gondii strains were maintained in human foreskin fibroblast (HFF) monolayers (ATCC, VA, USA) cultured in DMEM supplemented with 2% fetal bovine serum (Thermofisher Scientific, MA, USA), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, at 37 ℃ with 5% CO2.

Plasmid construction and generation of transgenic parasite lines

Locus-specific CRISPR plasmids were engineered using Q5 site-directed mutagenesis kit (NEB, MA, USA) to target distinct genomic regions of ent1 (pSAG1-CAS9-Δent1-sgRNA for ent1-CDS ablation, pSAG1-CAS9-iENT1-sgRNA for ent1 5’ UTR perturbation, and pSAG1-CAS9-TgENT1-mAID-sgRNA for ent1 3’ UTR integration) and CDS of aspartate transcarbamoylase (ATC) (pSAG1-CAS9-Δatc-sgRNA), as previously described (Shen et al. 2014). The recombinant plasmids containing corresponding homologous fragments were constructed using the ClonExpress one-step cloning kit (Vazyme Biotech, Nanjing, China). All primers used to amplify the fragments are listed in Table S1.

All transgenic strains were generated by CRISPR/CAS9-mediated homologous recombination, as previously described (Shen et al. 2017). Briefly, to make auxin-inducible degradation strain (TgENT1-mAID), the ent1 3’ UTR-targeting CRISPR plasmid and PCR amplicons containing ent1 CDS-mAID-HA-DHFR* were co-transfected into the RH-TIR1 parasites. Transfectants were selected with 1 µM pyrimethamine, single-cloned by limiting dilution, and validated by IFA assay and Western blot analysis. Similarly, the anhydrotetracycline (ATc)-mediated depletion strain (iENT1) and the ent1/atc knockout lines were constructed by co-transfecting, selecting, and verified by diagnostic PCRs.

Cell viability assay

The 50% cytotoxic concentrations (CC50) of NBMPR (MCE, Shanghai, China) targeting hENT1 on HFF cell line was measured with a cell proliferation assay and calculated by Prism 8.0.

Intracellular tachyzoite replication

The intracellular replication assays were used to assess the proliferation of T. gondii tachyzoites (RH, RHΔdho, and RHΔatc) under the indicated conditions. Freshly egressed tachyzoites were used to invade HFF monolayers seeded on coverslips in 24-well plates. Following 30 min of invasion at 37 ℃, noninvaded parasites were washed away with PBS, and the successfully invaded ones were subjected to pyrimidine sufficiency (250 µM uracil) and pyrimidine restriction (uracil-free condition), or nucleoside/nucleobase transport inhibition (5 µM or 10 µM NBMPR) and NBMPR-free control for 24 h. Subsequently, the samples were then fixed with 4% paraformaldehyde, and the number of parasites per parasitophorous vacuole (PV) was quantified by IFA. Statistical differences in Tg/PV numbers between sample groups were assessed by two-way analysis of variance (ANOVA), using GraphPad Prism 8.0. Each experiment was repeated three times independently.

IFA staining

For the intracellular replication assays, mouse anti-IMC1 polyclonal antibody (kindly provided by Dr Jin-Lei Wang, Lanzhou Veterinary Research Institute, 1:500 dilution) detected by Alexa FluoTM 488-conjugated goat anti-mouse IgG (Thermofisher Scientific; 1:1000 dilution), and rabbit anti-T. gondii polyclonal antibody (Abcam, Cambridge, UK; 1:1000 dilution) detected by Alexa FluorTM 594-conjugated goat anti-rabbit IgG (Thermofisher Scientific; 1:1000 dilution), were used to precisely quantify intracellular proliferation of tachyzoite. For strain identification of TgENT1-mAID, rabbit anti-HA monoclonal antibody (Abcam, Cambridge, UK; 1:100 dilution) and mouse anti-SAG1 monoclonal antibody (Thermofisher Scientific, 1:200 dilution) were used to visualize the expression and subcellular localization of TgENT1 in RH-TIR1 parasites.

Western blot analysis

Protein extracts from RH-TIR1 and TgENT1-mAID parasites were analyzed by immunoblot analysis following established protocols (Pan et al. 2019). Briefly, collected parasites were lysed in an ice-cold buffer containing 10 mM Tris-HCl, pH 6.8, 0.5% SDS (v/v), 10% glycerol (v/v), 1 mM EDTA, and protease inhibitor cocktail for 30 min, followed by heat denaturation for 10 min. Proteins were separated using SDS-PAGE and transferred to polyvinylidene fluoride membranes. The blots were blocked with 5% BSA, and then incubated with primary antibodies (rabbit anti-HA, 1:100 dilution; mouse anti-SAG1, 1:50 dilution) and secondary antibodies (HRP-conjugated goat anti-rabbit or anti-mouse IgG; Beyotime Biotechnology, China; 1:1000 dilution). Protein bands were imaged on a Tanon automatic chemiluminescence image analysis system according to the manufacturer’s protocol (Tanon, Shanghai, China).

Plaque assay

The overall growth of T. gondii tachyzoites was assessed by conventional plaque assays. Briefly, fresh confluent HFF monolayers in 6-well plates were infected with 200 parasites per well and incubated undisturbed at 37 °C for 7 days. Following infection, the cultures were supplemented with 250 µM uracil (for strains RHΔdho and RHΔatc) or with 500 µM indole-3-acetic acid (for strains RH-TIR1 and TgENT1-mAID), as indicated. The HFFs containing plaques were fixed with 4% paraformaldehyde (PA) and stained with 0.2% crystal violet solution. Statistical significance of relative plaque sizes was determined by one-way ANOVA.

Sequence alignment and phylogenetic analysis

Sequences of nucleoside and nucleobase transporters were retrieved from Uniprot (https://www.uniprot.org) and VEuPathDB (https://veupathdb.org/). The UniprotKB identifiers/Gene IDs are as follows, respectively: hENT1, Q99808/SLC29A1; hENT2, Q14542/SLC29A2; hENT3, Q9BZD2/SLC29A3; hENT4, Q7RTT9/SLC29A4; human concentrative nucleoside transporter 1 (hCNT1), O00337/SLC28A1; hCNT2, O43868/SLC28A2; hCNT3, Q9HAS3/SLC28A3; TgENT1, S8F309/TGME49_288540; TgENT3, A0A125YYW5/TGME49_233130; TgAT1, A0A125YNC9/TGME49_244440; TGGT1_359630, S7V3J7; Leishmania mexicana putative nucleoside transporter 1 (LmNT1A), E9AQB5/LMXM_15_1230; LmNT1B, E9AQB6/LMXM_15_1240; Trypanosoma brucei nucleobase/nucleoside transporter 8.1 (TbNBT1), Q8MUN2/NT8.1; Escherichia coli uracil permease (EcUraA), P0AGM7/UraA; Rattus norvegicus sodium-dependent nucleobase transporter 1 (rSNBT1), D2KX48/SLC23A4; Arabidopsis thaliana purine-uracil permease NCS1 (PLUTO), Q9LZD0; Saccharomyces cerevisiae uracil permease (ScFUR4), P05316/FUR4; Cryptococcus neoformans var. neoformans serotype D uracil transporter FurD (CnFurD), Q5K7G2/CNM02550; Aspergillus nidulans uracil transporter FurD (AnFurD), A6N844/FurD. Phylogenetic analysis was performed using MEGA 7. Nucleotide sequences were aligned with Clustal W, and evolutionary relationships were reconstructed via the neighbor-joining algorithm. Branch support was assessed with 1000 bootstrap replicates, and the Poisson-corrected genetic distances were calculated. The tree was drawn to scale, with branch lengths measuring the number of substitutions per site.

Molecular docking and molecular dynamics simulation

A comparative structural analysis of hENT1 (PDB ID: 6OB6; (Wright and Lee 2019) and the TgENT1 AlphaFold model (UniProt) was conducted in PyMOL 3.1. The analysis focused on the confidently predicted regions of TgENT1, defined by a pLDDT score greater than 70. NBMPR-proximal residues (< 6Å) in hENT1 were systematically mapped onto these conserved domains. Local root-mean-square deviation (RMSD) values were calculated for corresponding residues to evaluate the conservation of the functional site.

Molecular docking of NBMPR, nucleosides (uridine, adenosine) and nucleobases (adenine, uracil) to TgENT1 was performed using the Glide module (XP mode) within the Schrödinger suite. The top 10 poses from each ligand were subjected to induced-fit docking (IFD) for side-chain and backbone refinement. Their binding affinities were then re-evaluated by MM-GBSA, and the pose with the most favorable (lowest) MM-GBSA score was selected for subsequent molecular dynamics (MD) simulations. A 100-ns MD simulation was carried out using GROMACS, and the binding stability was assessed by performing MM-PBSA calculations on the last 10 ns of the production trajectory.

Statistical analysis

Statistical analyses were performed with GraphPad Prism 8.0, employing a two-way ANOVA (replication assay) or a one-way ANOVA (plaque assay), with details provided in the respective figure legends.

Results

Disruption of de novo pyrimidine biosynthesis in T. gondii renders exogenous uracil acquisition and transport essential for parasite proliferation

All six canonical enzymes comprising the de novo pyrimidine biosynthesis pathway have been molecularly characterized in T. gondii (Asai et al. 1983). Targeted disruption of CPSII, OPRT, OMPDC, or DHO results in severe uracil auxotrophy due to impaired UMP biosynthesis (Fox and Bzik 2002, 2010; Pan et al. 2024). To genetically dissect this pathway, we targeted the TGME49_291640 gene encoding aspartate transcarbamoylase (ATC), which catalyzes the second step of de novo pyrimidine synthesis (Fig. 1), and generated RHΔatc knockout mutants using CRISPR/CAS9-mediated gene replacement. Subsequently, the replication assays revealed that ATC deletion severely impaired intracellular tachyzoite proliferation, as evidenced by significantly reduced PV occupancy. Supplementation with uracil restored the growth of RHΔatc strain through the pyrimidine salvage pathway (Fig. 2A), consistent with previously observed rescue by uracil or high-dose uridine in the Δompdc genetic background (Fox and Bzik 2010). The plaque assay revealed that RHΔatc failed to form distinguishable plaques on host cell monolayers, unlike the parental RH strain. This growth restriction was reversible upon uracil supplementation, which restored plaque formation to wild-type levels (Fig. 2B, C). This reversibility suggests that ATC deletion impairs parasite growth in vitro by disrupting pyrimidine biosynthesis, a phenotype that mirrors deletions of other genes in the T. gondii de novo pyrimidine pathway (Fox and Bzik 2002, 2010; Pan et al. 2024). Similarly, DHO deletion recapitulated the proliferative defect that was rescued by uracil. Altogether, targeted ablation of de novo pyrimidine biosynthesis genes uniformly generates obligate uracil auxotrophs in T. gondii type I parasites. This genetic impairment is compensated by a critical pyrimidine salvage pathway, which is essential for sustaining parasite viability and growth.

Fig. 2.

Fig. 2

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hENT1 inhibitor NBMPR impairs growth of pyrimidine auxotrophic parasites by blocking uracil transport. (A) Intracellular replication assay comparing parasite proliferation under the indicated condition (± uracil). Following synchronized invasion of HFF cells, parasites were allowed to proliferate for 24 h. The number of parasites (1, 2, 4, 8, ≥ 16) per parasitophorous vacuole was then quantified by IFA. Data are represented as the means ± SEM from three independent experiments. The statistical significance of differences in Tg/PV numbers across groups was assessed using two-way ANOVA, *0.01 ≤ p < 0.05, **0.001 ≤ p < 0.01, ***p < 0.001, NS: not significant. (B) Plaque assay comparing the growth of RHΔdho and RHΔatc parasites with or without 250 µM uracil supplementation to that of parental strain RH. Two hundred purified tachyzoites were used to infect HFFs for 7 days and then stained by 0.1% crystal violet. (C) Relative plaque sizes (calculated by Photoshop) formed by RHΔdho and RHΔatc strains versus RH strain from panel B. Data represent means ± SEM. Statistical significance was determined by one-way ANOVA, ***p < 0.001. (D) Model of nucleoside/nucleobase transport dynamics upon T. gondii infection in HFF cells. The proposed two-step transport mechanism involves: (1) host nutrient uptake across the plasma membrane and into intracellular organelles, and (2) subsequent acquisition by T. gondii. Each step is mediated by distinct transporter families. N, nucleus; Mi, mitochondrion. (E) Human SLC29 (hENTs) and SLC28 (hCNTs) transporters: cellular localization, substrate selectivity, and specificity. Apparent Km values were determined in expression systems using cell lines and Xenopus laevis oocytes (Pastor-Anglada et al. 2018). (F) Intracellular replication of parasites with (+) or without 10 µM NBMPR (-) for 24 h. Data represent means ± SEM of three independent experiments, and were analyzed by two-way ANOVA, *0.01 ≤ p < 0.05, **0.001 ≤ p < 0.01, ***p < 0.001. (G) Assessment of intracellular replication of parasites treated with 5 µM NBMPR (+) versus untreated controls (-) over 24 h. Values are means ± SEM of n = 3 independent experiments. Differences between means were analyzed by two-way ANOVA, *0.01 ≤ p < 0.05, **0.001 ≤ p < 0.01, ***p < 0.001

NBMPR-mediated transport inhibition of uracil in T. gondii pyrimidine auxotrophic parasites

Based on the collective evidence from this and previous studies (Fox and Bzik 2010; Fox and Bzik 2015) demonstrating efficient parasite rescue by uracil or uridine in pyrimidine auxotrophic strains, we therefore propose a two-step model for nucleoside/nucleobase scavenging in T. gondii: (i) host cell uptake via mammalian nucleoside/nucleobase transporters, followed by (ii) parasite-specific acquisition from the host cytosol (Fig. 2D).

In humans, membrane transport of nucleosides or nucleobases is mediated by two genetically distinct protein families: the equilibrative and concentrative nucleoside transporter (ENTs and CNTs) families (Fig. 2E). ENTs typically function as facilitative uniporters, mediating the basolateral efflux of nucleosides and nucleobases into the bloodstream, whereas CNTs are sodium-dependent secondary active symporters primarily located at the apical side of enterocytes (Pastor-Anglada et al. 2018). Among these, hENT1 and hENT2 are key mediators responsible for the membrane transport of various nucleosides (e.g., adenosine, uridine, cytidine) and nucleobases (e.g., adenine, uracil), and demonstrating versatile substrate specificity (Pastor-Anglada et al. 2018) (Fig. 2E). The two transporters display a dramatic difference in sensitivity to the purine nucleoside analogue Nitrobenzylthioinosine (NBMPR): hENT1 is potently inhibited with high affinity (IC50 = 0.4 ± 0.1 nM), whereas hENT2 is relatively resistant (IC50 = 2.8 ± 0.3 µM) (Ward et al. 2000). We therefore seek to investigate whether blocking these transporters with NBMPR disrupts uracil transport in the pyrimidine auxotroph of T. gondii. Initial cytotoxicity assessment confirmed that NBMPR was well tolerated by host cells (CC50 > 200 µM in HFF cells; Fig. S1), in agreement with previous findings (el Kouni et al. 1999). We subsequently evaluated the effects of NBMPR treatment on the intracellular proliferation of T. gondii. Treatment with 10 µM NBMPR, which blocks hENT1 and partially inhibits hENT2, caused significant growth inhibition across RHΔatc, RHΔdho, and wild-type RH strains relative to the NBMPR-free control (Fig. 2F). Uracil supplementation did not rescue the growth inhibition observed in NBMPR-treated RHΔatc and RHΔdho parasites (Fig. 2F). Based on a reported 50% inhibitory concentration (IC50) of 10.2 µM for NBMPR against T. gondii (with 5 µM achieving approximately 14% inhibition) (el Kouni et al. 1999; Elati et al. 2023), we selected the lower 5 µM concentration to minimize confounding effects from direct parasite toxicity. At this concentration, NBMPR had no significant effect on the wild-type RH strain. Strikingly, however, it still effectively inhibited the intracellular proliferation of both RHΔatc and RHΔdho mutants, even with uracil rescue (Fig. 2G). Collectively, these results confirm that NBMPR selectively impairs the growth of pyrimidine-auxotrophic parasites by inhibiting uracil uptake, through its targeting of nucleoside/nucleobase transport in T. gondii-infected HFFs.

The evolutionary conservation and structural similarities of TgENT1 with nucleoside/nucleobase transporters

Beyond the host’s intrinsic nucleoside/nucleobase uptake via hENT1 and hENT2, we propose that T. gondii exploits a functionally similar yet genetically independent transporter system to directly scavenge these nutrients from the host cytosol (Fig. 2D). To identify potential nucleoside/nucleobase transporters in T. gondii, we conducted BLAST searches against the ToxoDB (https://toxodb.org/toxo/app/) using hENT1 as a query. BLASTp analysis identified three candidate nucleoside transporters (TGME49_288540, TGME49_233130, TGGT1_359630) and the known adenosine transporter TgAT1 (TGME49_244440) (Chiang et al. 1999) as homologs of hENT1. Among these, TGME49_288540 and TGME49_233130 have recently been designated as TgENT1 and TgENT3, respectively (Messina et al. 2025). Phylogenetic reconstruction revealed that TgENT1 exhibited closer evolutionary conservation with hENTs than with hCNTs (Fig. 3A), suggesting a dual transport capacity for both nucleosides and nucleobases in T. gondii (Fig. 2E). hENT1 and hENT2, which possess 11 transmembrane domains (TMs), belong to a conserved family of integral membrane proteins (Yao et al. 2002). Sequence alignment indicated that TgENT1 similarly contains 11 TMs and retained the glycosylated loop between TM1 and TM2 (Fig. 3B), consistent with the human proteins. In contrast to its human counterparts, TgENT1 exhibited extended regions in both TM3 and TM5, along with a longer cytoplasmic loop between TM6 and TM7 (Fig. 3B). We further compared TgENT1 with known uracil transporters from diverse lineages. It showed limited sequence identity with its apicomplexan orthologs LmNT1 (25.7%) and TbNBT1 (25.9%). Phylogenetically, TgENT1 formed a distant clade, sharing only 16.9–22.8% identity with confirmed bacterial, plant, and fungal transporters (Fig. 3C). Consistently, direct sequence alignment confirmed its low homology with key transporters including LmNT1, TbNBT1, and EcUraA (Fig. 3D).

Fig. 3.

Fig. 3

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Phylogenetic analyses of nucleoside/nucleobase transporter candidates in T. gondii. (A) Phylogenetic analysis of T. gondii nucleoside/nucleobase transporters with hENTs and hCNTs. Evolutionary relationships have been done in MEGA 7 using the neighbor-joining algorithm. (B) Multiple sequence alignment of TgENT1 and hENTs. The transmembrane domains (TMs), glycosylated loop and cytoplasmic loop are indicated based on the known features of hENT1 and hENT2 (Yao et al. 2002). (C) Phylogenetic placement of T. gondii nucleoside/nucleobase transporters within a broader context of uracil transporters from diverse taxa. (D) Multiple sequence alignment of TgENT1 and uracil transporters. All sequences of nucleoside and nucleobase transporters are retrieved from Uniprot (https://www.uniprot.org) and VEuPathDB (https://veupathdb.org/)

Given that hENT1 has been co-crystallized in complex with NBMPR (Wright and Lee 2019), we used its structure as a template to predict the TgENT1 structure using AlphaFold. Global structural superposition revealed moderate conservation between TgENT1 and hENT1 (RMSD = 3.213 Å) (Fig. 4A). Strikingly, the NBMPR-binding residues maintained remarkable structural conservation (RMSD = 0.163 Å) (Fig. 4B). Following IFD refinement, the NBMPR-TgENT1 complex achieved a significantly improved IFD Score of −15907.28, coupled with a robust MM-GBSA binding free energy of −74.58 kcal/mol, indicating thermodynamically stabilized binding compared to the initial docking pose with a MM-GBSA of −59.99 kcal/mol (Fig. 4C, Table S2). Throughout the ‌100 ns molecular dynamics trajectory‌, the ligand maintained stable positioning without significant drift. The final 10 ns ensemble yielded a binding free energy of −16.48 kcal/mol‌, corresponding to an inhibition constant (Ki) of ‌8.33 × 10− 7 µM‌, further confirming high-affinity binding of NBMPR-TgENT1 (Table S3). To assess whether TgENT1 functions as a broad-specificity nucleoside/nucleobase transporter similar to hENT1, we performed molecular docking analyses with representative substrates: nucleosides (uridine and adenosine) and nucleobases (adenine and uracil) (Fig. 4D). The calculated binding affinities (MM-GBSA) revealed strong interactions for nucleosides (−45.3 and − 44.8 kcal/mol for uridine and adenosine, respectively) and moderate interactions for nucleobases (approximately − 28 kcal/mol for both adenine and uracil) (Fig. 4D, Table S4). These results indicate that, like hENT1, TgENT1 likely functions as a nucleoside/nucleobase transporter involved in purine and pyrimidine acquisition in T. gondii.

Fig. 4.

Fig. 4

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Structure prediction and molecular docking analysis of TgENT1. (A) Whole-structure superposition analysis of hENT1 and TgENT1. The structure of hENT1 (PDB ID: 6OB6; Uniprot ID: Q99808; green) and TgENT1 (AlphaFold-predicted structure; Uniprot ID: S8F309; light blue) is performed using the PyMOL3.1. (B) Comparative structural analysis of hENT1 and TgENT1, focusing on the NBMPR-binding residues within 6Å. (C) The predicted binding pose of NBMPR in TgENT1, obtained using the Schrödinger suite, with a 2D interaction diagram showing key contacts. (D) Predicted binding poses of TgENT1 with nucleoside/nucleobase substrates: uridine, adenosine, adenine, and uracil

Subcellular localization analysis and the genetic manipulation of TgENT1

Considering the characteristics of TgENT1 in nucleoside/nucleobase transport, we first sought to investigate its functional contribution to parasite growth through targeted gene deletion. Using CRISPR/CAS9-mediated homologous recombination, we replaced the coding sequence (CDS) of ent1 with a pyrimethamine-resistant cassette DHFR* to generate a knockout strain (Fig. S2A). Although viable mutants were successfully isolated, diagnostic PCRs failed to confirm correct DHFR* integration, as evidenced by the absence of the expected PCR1 products (Fig. S2B). This suggests that TgENT1 performs an essential function, consistence with its relatively low phenotype score (−3.68). Subsequently, an anhydrotetracycline (ATc)-inducible knockdown system (iENT1) was used to deplete TgENT1 expression by a promoter replacement strategy (Fig. S2C). However, diagnostic PCR screening across the homology arms yielded no positive clones throughout multiple parasite generations (G3 and G7) (Fig. S2D). Therefore, a mini-AID (auxin inducible degron) tag (mAID) and a HA tag were simultaneously fused to the C-terminus of TgENT1 in parasites stably expressing the auxin receptor transport inhibitor response 1 (RH-TIR1) (Fig. 5A). Immunofluorescence analysis revealed that TgENT1-mAID-HA exhibited a punctate pattern within the parasite cytosol and did not colocalize with the plasma membrane marker SAG1 (Fig. 5B). Subsequently, we assessed the downregulation of TgENT1 by Western blot analysis. Administration of indole-3-acetic acid (IAA), a natural auxin, did not decrease TgENT1 protein levels during the 2–18 h treatment (Fig. S3A). Consistent with these findings, plaque assays revealed no significant impairment in parasite growth (Fig. S3B). Collectively, our inability to generate either a knockout or a conditional depletion of TgENT1 strain implies that this protein is essential for parasite growth, which is consistent with and strongly corroborated by previous evidence (Messina et al. 2025).

Fig. 5.

Fig. 5

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Localization analysis of TgENT1. (A) Schematic illustration of endogenous C-terminal tagging of TgENT1 with a mAID-HA tag via CRISPR/Cas9-mediated homologous recombination. (B) Immunofluorescent staining using mouse-derived anti-SAG1 and rabbit-derived anti-HA antibodies to assess the expression and subcellular localization of TgENT1 in TgENT1-mAID parasites. Scale bar, 5 μm

Discussion

With the remarkably broad host range, T. gondii stands as one of the most successful parasitic pathogens. To thrive across diverse host environments, the parasite has evolved complex mechanisms of intracellular parasitism. Particularly noteworthy is its metabolic flexibility: the sophisticated ability to balance de novo metabolite synthesis with acquisition of host-derived nutrients, that is fundamental to T. gondii persistence and pathogenesis. The nucleotide biosynthesis pathways are critical for rapid parasite proliferation as it generates UMP and IMP (inosine 5’-monophosphate), the essential precursors for all nucleotides (Fig. 1). Our prior work demonstrated that DHO-mediated de novo pyrimidine biosynthesis is critical for robust parasite proliferation. Significantly, exogenous uracil supplementation effectively restores growth in DHO-deficient parasites (Pan et al. 2024), uncovering a compensatory nucleobase salvage pathway that maintains pyrimidine homeostasis. In the present study, such rescue was abolished under NBMPR treatment, suggesting the involvement of a previously unrecognized transporter system for parasite-specific nucleoside/nucleobase acquisition from the host cytosol. Functional characterization of TgENT1 further indicates that this nucleoside/nucleobase transporter is essential for parasite proliferation. These findings support a novel anti-toxoplasmosis strategy that targets the acquisition of both nucleosides and nucleobases in T. gondii.

Given the critical role in mediating adenosine and nucleoside analog uptake, hENT1 has emerged as a promising pharmacological target for developing non-addictive pain medications (Wright and Lee 2019). To probe this system, we employed NBMPR, a potent and specific inhibitor, to selectively block hENT1 and partially inhibit hENT2. Treatment with 5 or 10 µM NBMPR effectively suppressed host uracil uptake, thereby impaired the growth of pyrimidine-auxotrophic T. gondii tachyzoites that rely exclusively on salvage pathways (Fig. 2F, G). During chronic infection, we previously demonstrated that DHO-deficient parasites formed normal numbers of brain cysts in mice, albeit with reduced cyst size (Pan et al. 2024). Since host-derived UMP is inaccessible to intracellular parasites (Fox and Bzik 2010), these findings provide strong evidence that PruΔdho bradyzoites compensate for pyrimidine auxotrophy by scavenging host pyrimidine pools to fulfill their metabolic requirements and sustain cyst persistence. Consequently, the dual targeting strategy, combining NBMPR-mediated inhibition of host ENT1 with DHO/ATC inhibitors that block T. gondii de novo pyrimidine biosynthesis, represents a promising therapeutic approach. It would be particularly effective against bradyzoites, as pharmacological blockade of ENT1 would exhaust host cytosolic pyrimidine pools, thereby depriving the pyrimidine auxotroph of critical salvage pathway substrates required for persistence. Moreover, recent development of JH-ENT-01, a rationally designed hybrid incorporating dilazep (a non-nucleoside vasodilator) and NBMPR pharmacophores (Wright and Lee 2019; Wright et al. 2024), provides a template for developing hENT1-targeted therapeutics with enhanced binding specificity.

Based on the evolutionary conservation and structural homology to hENT1, TgENT1 was identified as the primary candidate transporter for nucleosides and nucleobases in T. gondii, although potential contributions from TgENT3 and TGGT1_359630 cannot be excluded. While a previous study suggested that NBMPR inhibits wild-type T. gondii through its metabolic products (el Kouni et al. 1999), our findings demonstrate that it directly and simultaneously targets both hENT1 and TgENT1 (Fig. 4B, C). Given that T. gondii is a purine auxotroph and hENT1 is crucial for adenosine transport, the observed growth inhibition of the wild-type RH strain by 10 µM NBMPR is best explained by the dual targeting of hENT1 and TgENT1, which blocks the parasite’s salvage of essential exogenous purines such as adenosine and hypoxanthine. To identify the specific nutrients that interact with TgENT1, we attempted to recombinantly express a His-tagged version of the transporter. However, the resulting insoluble protein precluded direct biochemical characterization of its ligand binding and transport functions. Critical next steps include achieving soluble TgENT1 production through fusion partners like GST or MBP, which would permit direct measurement of transport kinetics. Subsequently, successful expression would open the door to crystallizing TgENT1 in complex with nucleosides/nucleobases, revealing the atomic-level details of substrate binding to rationally guide future inhibitor design.

Although our study faced technical limitations that precluded the genetic disruption of TgENT1, its essential role is strongly supported by independent work. Messina et al. demonstrated that depleting TgENT1 in a conditional knockdown strain led to complete growth arrest, and a purine-starvation-like transcriptional profile (Messina et al. 2025). Their findings directly validate our functional prediction that TgENT1, as a dual nucleoside/nucleobase transporter, is critical for parasite proliferation. Contrary to the typical plasma membrane localization of many transporters, we found that TgENT1 exhibits a distinct punctate pattern within the parasite cytosol (Fig. 5B). This observation aligns with the findings of Messina et al., who identified TgENT1 in the plant-like vacuolar compartment (PLVAC) and showed that its loss causes PLVAC swelling. Their work indicates that T. gondii strategically acquires purines by degrading host-derived or recycling its own nucleic acids within the acidic, digestive environment of the PLVAC (Messina et al. 2025). This strategy enables the efficient salvage and utilization of nucleosides/nucleobases to meet the high nucleotide demands of rapid parasite proliferation, thus providing a rationale for the non-canonical intracellular localization of TgENT1. To further validate TgENT1’s function, a powerful approach would be to combine pyrimidine auxotrophic T. gondii strains expressing endogenously tagged TgENT1 with stable, biotinylated uracil analogs. Their simultaneous detection via streptavidin staining and epitope-specific immunofluorescence would thereby enable direct spatiotemporal tracking of uracil localization alongside TgENT1 trafficking dynamics across replicative stages.

Despite functional characterization of uracil transporters in bacteria (Lu et al. 2011), fungi (Grossmann et al. 2008), mammals (Yamamoto et al. 2010; Yao et al. 2011), and plant (Witz et al. 2012), as well as in protozoa (Alzahrani et al. 2017; Natto et al. 2021), the research on uracil uptake and transport in T. gondii is limited. Previous studies using radiolabeled transport assays (3H-uridine and 3H-uracil) demonstrated that T. gondii tachyzoites took up both uridine and uracil with similar high affinity, suggesting they shared a common transporter designated as uridine/uracil transporter 1 (TgUUT1) (Elati et al. 2023). Strikingly, TgUUT1 differs from characterized uracil transporters in other protozoa, where uracil transport is mediated by separate, dedicated transporters (Natto et al. 2021; Aldfer et al. 2022). Furthermore, while TgUUT1 mediates uridine/uracil transport in T. gondii, its L. mexicana ortholog has evolved high-affinity adenosine transport activity (Alzahrani et al. 2017). In this study, phylogenetic analysis shows that TgENT1 forms a distant clade from canonical uracil transporters. It shares significant homology with hENT1 (a major facilitator of adenosine uptake) and displays interactions with uridine and uracil. The functional similarity between TgENT1 and TgUUT1 prompts future work to elucidate their specific roles and potential interplay. Furthermore, it remains unclear whether the known high-affinity transporters TgNBT1 (nucleobases) and AT2 (adenosine) (De Koning et al. 2003) are functionally associated with TgENT1.

Conclusion

Our integrated analysis identifies a hierarchical transport cascade reliant on both TgENT1 and host hENT1 as a functionally pathway for parasite nucleotide acquisition. This study unveils TgENT1 as a novel target and, more importantly, provides the rationale for a dual-targeting strategy aimed at simultaneously disrupting the transport functions of both the parasite and its host.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1.1MB, docx)

Acknowledgements

We gratefully acknowledge Dr. Jin-Lei Wang for generously supplying the mouse anti-IMC1 antibody.

Author contributions

MP designed the research; MP and SQT performed the experiments, acquired the data; MP analyzed the data and drafted the first version of the manuscript; CCG, SJF, HXH, and MMB participated in the implementation of this work; BS and SYH critically revised the manuscript. All authors reviewed the manuscript.

Funding

This work was funded by the Natural Science Foundation of Jiangsu Province (BK20230579), the National Natural Science Foundation of China (32503064), and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Data availability

The data that support the findings of this study are available in the supplementary material of this article.

Declarations

Ethics declaration

Not applicable.

Consent for publication

All authors gave their consent before submitting this work.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Ming Pan, Email: panming@yzu.edu.cn.

Si-Yang Huang, Email: siyang.huang@hotmail.com.

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Supplementary Materials

Supplementary Material 1 (1.1MB, docx)

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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