Toxoplasma gondii Archives - Center for Tropical and Emerging Global Diseases

Calcium transfer from the ER to other organelles for optimal signaling in Toxoplasma gondii

Donna Huber on November 12, 2025

Hypothetical model showing Ca2+ entry through two different types of Ca2+ channels, uptake by T. gondii sarco/endoplasmic reticulum Ca2+- ATPase (TgSERCA) into the endoplasmic reticulum (ER) and distribution to the other organelles via transfer from the ER to the mitochondria, Plant-Like Vacuolar Compartment (PLVAC), and apicoplast.

Ca2+ signaling in cells begins with the opening of Ca2+ channels in either the plasma membrane (PM) or endoplasmic reticulum (ER), leading to a sharp increase in the physiologically low (<100 nM) cytosolic Ca2+ level. The temporal and spatial regulation of Ca²+ is crucial for the precise activation of key biological processes. In the apicomplexan parasite Toxoplasma gondii, which infects approximately one-third of the global population, Ca²+ signaling governs essential aspects of the parasite’s infection cycle. T. gondii relies on Ca²+ signals to regulate pathogenic traits, with several Ca²+-signaling components playing critical roles. Ca2+ entry from the extracellular environment has been demonstrated in T. gondii for both, extracellular parasites, exposed to high Ca2+, and intracellular parasites, which acquire Ca²+ from host cells during host Ca²+ signaling events. Active egress, an essential step of the parasite’s infection cycle, is preceded by a large increase in cytosolic Ca2+, most likely initiated by release from intracellular stores. However, extracellular Ca2+ is also necessary to reach a cytosolic Ca2+ threshold required for timely egress. In this study, we investigated the mechanism of intracellular Ca²+ store replenishment and identified a central role for the SERCA-Ca2+-ATPase in maintaining Ca²+ homeostasis within the ER and in other organelles. We demonstrate mitochondrial Ca2+ uptake, which occurs by transfer of Ca2+ from the ER, likely through membrane contact sites. Our findings suggest that the T. gondii ER plays a key role in sequestering and redistributing Ca²+ to intracellular organelles following Ca²+ influx at the PM.

Zhu-Hong Li, Beejan Asady, Le Chang, Myriam Andrea Hortua Triana, Catherine Li, Isabelle Coppens, Silvia N J Moreno. Elife. 2025 Nov 12:13:RP101894. doi: 10.7554/eLife.101894.

O-fucosylation affects abundance but not localization of select nucleocytoplasmic proteins in Toxoplasma gondii

Donna Huber on September 1, 2025

Figure one from the paper — Analysis of epitope-tagged GPN1 in cells. A) Schematic of GPN1 protein indicating conserved domains.

Toxoplasma gondii is a highly successful intracellular mammalian and avian pathogen that must adapt to a wide range of intracellular and extracellular environments. A mechanism that may support this is the modification of hydroxyamino acid rich sequences of nucleocytoplasmic proteins with O-fucose. O-fucosylation of possibly hundreds of proteins is mediated by a single highly conserved nucleocytoplasmic enzyme. Deletion of the SPY O-fucosyltransferase gene is tolerated but inhibits parasite proliferation in fibroblasts and their accumulation in mouse brains. A prior ectopic expression study suggested that O-fucose is required to detect proteins considered essential. To distinguish whether the SPY requirement was specific to the method or for protein expression per se, GPN1, an RNA polymerase chaperone, was epitope-tagged at its endogenous locus in both normal and SPYΔ strains. GPN1 was shown to be substantially and quantitatively O-fucosylated and exhibited a modest 24% reduction in level in SPYΔ cells. Proteomic analysis of its interactome indicated that fucosylation did not affect its association with RNA polymerase subunits. GPN1 was mostly cytoplasmic based on super-resolution immunofluorescence microscopy, and this localization was not affected by O-Fuc. A fusion of its O-fucosylated serine-rich domain to yellow fluorescent protein behaved similarly. In comparison, the abundance of a Zn-finger containing protein also depended on SPY, whereas the abundance and localization of ERK7 were not affected nor were levels of two other proteins. Thus O-fucose directly but modestly promotes the accumulation of select targets, but it does not enforce their localization in nuclear assemblies that are highlighted by immunofluorescence studies.

Megna Tiwari, Elisabet Gas-Pascual, Janice Teal-Urquides, John Samuelson, Christopher M West. Glycobiology. 2025 Sep 1:cwaf051. doi: 10.1093/glycob/cwaf051.

Activity of Antibacterial/Antifungal Compounds against the Protozoan Parasite, Toxoplasma gondii

Donna Huber on August 28, 2025

We investigated the antiparasitic activity of several antimicrobial drug leads against Toxoplasma gondii tachyzoites and, in one case, bradyzoites. Carbazole and phenylthiazole aminoguanidine anti-infectives, originally developed as antibacterial and antifungal agents, showed potent activity, with IC₅₀ values as low as 2 μM. This potency was comparable to that observed with the tuberculosis drug candidate SQ109 and a series of its analogs. Notably, SQ109 also significantly reduced the viability of in vivo-derived bradyzoites. All compounds acted, at least in part, as protonophore uncouplers by collapsing the ΔpH component of the proton motive force. Furthermore, SQ109 and the tetrahydrocarbazole (THCz) compounds disrupted the mitochondrial membrane potential in T. gondii tachyzoites. While SQ109 is known to activate macrophages to an M1 phenotype, we observed no significant difference in its activity against T. gondii grown in fibroblasts versus macrophages, likely due to the parasite’s residence within the protective parasitophorous vacuole. We also examined correlations between compound activity against the yeast Saccharomyces cerevisiae, and the bacterium Mycobacterium smegmatis, finding significant correlations between the collapse of the proton motive force and antiproliferative activity. Taken together, our findings underscore the potential of these antimicrobial agents as promising leads for the development of new antiparasitic therapies against T. gondii.

Davinder Singh, Melissa A Sleda, Satish R Malwal, Akanksha M Pandey, Yiyuan Chen, Ruijie Zhou, Feyisola Adewole, Katie Sadowska, Oluseye K Onajole, Silvia N J Moreno, Eric Oldfield. ACS Infect Dis. 2025 Aug 28. doi: 10.1021/acsinfecdis.5c00609.

The contribution of the Golgi and the endoplasmic reticulum to calcium and pH homeostasis in Toxoplasma gondii

Donna Huber on March 3, 2025

TgCAXL1 localizes to the Golgi apparatus and the endoplasmic reticulum.

The cytosolic Ca²⁺ concentration of all cells is highly regulated demanding the coordinated operation of Ca2+ pumps, channels, exchangers and binding proteins. In the protozoan parasite Toxoplasma gondii calcium homeostasis, essential for signaling, governs critical virulence traits. However, the identity of most molecular players involved in signaling and homeostasis in T. gondii are unknown or poorly characterized. In this work we studied a putative calcium proton exchanger, TgGT1_319550 (TgCAXL1), which belongs to a family of Ca²⁺/proton exchangers that localize to the Golgi apparatus. We localized TgCAXL1 to the Golgi and the endoplasmic reticulum (ER) of T. gondii and validated its role as a Ca²⁺/proton exchanger by yeast complementation. Characterization of a knock-out mutant for TgCAXL1 (Δcaxl) underscored the role of TgCAXL1 in Ca²⁺ storage by the ER and acidic stores, most likely the Golgi. Most interestingly, TgCAXL1 function is linked to the Ca²⁺ pumping activity of the Sarcoplasmic Reticulum Ca²⁺-ATPase (TgSERCA). TgCAXL1 functions in cytosolic pH regulation and recovery from acidic stress. Our data showed for the first time the role of the Golgi in storing and modulating Ca²⁺ signaling in T. gondii and the potential link between pH regulation and TgSERCA activity, which is essential for filling intracellular stores with Ca²⁺.

Abigail Calixto, Katherine Moen, Silvia Nj Moreno. J Biol Chem.. 2025 Mar 3:108372. doi: 10.1016/j.jbc.2025.108372.

mSphere of Influence: Lighting up organellar communication in protozoan parasites

Donna Huber on February 6, 2025

Diego Huet works in molecular parasitology, focusing on the organellar biology of Toxoplasma gondii. In this mSphere of Influence article, he reflects on how the article “Efficient proximity labeling in living cells and organisms with turboID” (Branon et al., 2018) impacted his research and the strategies used to dissect inter-organellar interactions in T. gondii.

Diego Huet. mSphere. 2025 Feb 6:e0057424. doi: 10.1128/msphere.00574-24.

The Toxoplasma gondii homolog of ATPase inhibitory factor 1 is critical for mitochondrial cristae maintenance and stress response

Donna Huber on November 27, 2024

Generation of the IF1Ty, IF1KO, and IF1Over strains.

The production of energy in the form of ATP by the mitochondrial ATP synthase must be tightly controlled. One well-conserved form of regulation is mediated via ATPase inhibitory factor 1 (IF1), which governs ATP synthase activity and gene expression patterns through a cytoprotective process known as mitohormesis. In apicomplexans, the processes regulating ATP synthase activity are not fully elucidated. Using the model apicomplexan Toxoplasma gondii, we found that knockout and overexpression of TgIF1, the structural homolog of IF1, significantly affected gene expression. Additionally, TgIF1 overexpression resulted in the formation of a stable TgIF1 oligomer and increased the presence of higher order ATP synthase oligomers. We also show that parasites lacking TgIF1 exhibit reduced mitochondrial cristae density, and that while TgIF1 levels do not affect growth in conventional culture conditions, they are crucial for parasite survival under hypoxia. Interestingly, TgIF1 overexpression enhances recovery from oxidative stress, suggesting a mitohormetic function. In summary, while TgIF1 does not appear to play a role in ATP synthase regulation under conventional growth conditions, our work uncovers its potential role in adapting to the stressors faced by T. gondii and other apicomplexans throughout their intricate life cycles.

Madelaine M Usey, Anthony A Ruberto, Kaelynn V Parker, Diego Huet. Mol Biol Cell. 2024 Nov 27:mbcE24080344. doi: 10.1091/mbc.E24-08-0344.

Lipophilic bisphosphonates reduced cyst burden and ameliorated hyperactivity of mice chronically infected with Toxoplasma gondii

Donna Huber on October 10, 2024

Fig 1 Lipophilic bisphosphonates inhibited the viability of in vitro differentiated bradyzoites.

The current treatments for toxoplasmosis are only active against fast-growing tachyzoites, present in acute infections, with little effect on slow-growing bradyzoites within tissue cysts, present in latent chronic infections. The mitochondrion of Toxoplasma gondii is essential for its survival, and one of the major anti-parasitic drugs, atovaquone, inhibits the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase site. Coenzyme Q (also known as ubiquinone [UQ]) consists of a quinone head and a lipophilic, isoprenoid tail that anchors UQ to membranes. The synthesis of the isoprenoid unit is essential for cell growth and is inhibited by lipophilic bisphosphonates, which inhibit the parasite growth. In this work, we investigated the effect of lipophilic bisphosphonates on the chronic stages of T. gondii. We discovered that three lipophilic bisphosphonates (BPH-1218, BPH-1236, and BPH-1238), effective for the acute infection, were also effective in controlling the development of chronic stages. We showed effectiveness by testing them against in vitro cysts and in vivo derived tissue cysts and, most importantly, these compounds reduced the cyst burden in the brains of chronically infected mice. We monitored the activity of infected mice non-invasively and continuously with a novel device termed the CageDot. A decrease in activity accompanied the acute phase, but mice recovered to normal activity and showed signs of hyperactivity when the chronic infection was established. Moreover, treatment with atovaquone or BPH-1218 ameliorated the hyperactivity observed during the chronic infection.IMPORTANCETreatment for toxoplasmosis is challenged by a lack of effective drugs to eradicate the chronic stages. Most of the drugs currently used are poorly distributed to the central nervous system, and they trigger allergic reactions in a large number of patients. There is a compelling need for safe and effective treatments for toxoplasmosis. Bisphosphonates (BPs) are analogs of inorganic pyrophosphate and are used for the treatment of bone disorders. BPs target the isoprenoid pathway and are effective against several experimental parasitic infections. Some lipophilic BPs can specifically inhibit the mitochondrial activity of Toxoplasma gondii by interfering with the mechanism by which ubiquinone is inserted into the inner mitochondrial membrane. In this work, we present the effect of three lipophilic BPs against T. gondii chronic stages. We also present a new strategy for the monitoring of animal activity during disease and treatment that is non-invasive and continuous.

Melissa A Sleda, Zaid F Pitafi, WenZhan Song, Eric Oldfield, Silvia N J Moreno. mBio. 2024 Oct 10:e0175624. doi: 10.1128/mbio.01756-24

The GPI sidechain of Toxoplasma gondii inhibits parasite pathogenesis

Donna Huber on September 20, 2024

Fig 5 Immunofluorescence analysis of PIGJ-3×HA shows its localization both inside and outside the rER.

Glycosylphosphatidylinositols (GPIs) are highly conserved anchors for eukaryotic cell surface proteins. The apicomplexan parasite, Toxoplasma gondii, is a widespread intracellular parasite of warm-blooded animals whose plasma membrane is covered with GPI-anchored proteins, and free GPIs called GIPLs. While the glycan portion is conserved, species differ in sidechains added to the triple mannose core. The functional significance of the Glcα1,4GalNAcβ1- sidechain reported in Toxoplasma gondii has remained largely unknown without understanding its biosynthesis. Here we identify and disrupt two glycosyltransferase genes and confirm their respective roles by serology and mass spectrometry. Parasites lacking the sidechain on account of deletion of the first glycosyltransferase, PIGJ, exhibit increased virulence during primary and secondary infections, suggesting it is an important pathogenesis factor. Cytokine responses, antibody recognition of GPI-anchored SAGs, and complement binding to PIGJ mutants are intact. By contrast, the scavenger receptor CD36 shows enhanced binding to PIGJ mutants, potentially explaining a subtle tropism for macrophages detected early in infection. Galectin-3, which binds GIPLs, exhibits an enhancement of binding to PIGJ mutants, and the protection of galectin-3 knockout mice from lethality suggests that Δpigj parasite virulence in this context is sidechain dependent. Parasite numbers are not affected by Δpigj early in the infection in wild-type mice, suggesting a breakdown of tolerance. However, increased tissue cysts in the brains of mice infected with Δpigj parasites indicate an advantage over wild-type strains. Thus, the GPI sidechain of T. gondii plays a crucial and diverse role in regulating disease outcomes in the infected host.IMPORTANCEThe functional significance of sidechain modifications to the glycosylphosphatidylinositol (GPI) anchor in parasites has yet to be determined because the glycosyltransferases responsible for these modifications have not been identified. Here we present identification and characterization of both Toxoplasmsa gondii GPI sidechain-modifying glycosyltransferases. Removal of the glycosyltransferase that adds the first GalNAc to the sidechain results in parasites without a sidechain on the GPI, and increased host susceptibility to infection. Loss of the second glycosyltransferase results in a sidechain with GalNAc alone, and no glucose added, and has negligible effect on disease outcomes. This indicates GPI sidechains are fundamental to host-parasite interactions.

Julia A Alvarez, Elisabet Gas-Pascual, Sahil Malhi, Juan C Sánchez-Arcila, Ferdinand Ngale Njume, Hanke van der Wel, Yanlin Zhao, Laura García-López, Gabriella Ceron, Jasmine Posada, Scott P Souza, George S Yap, Christopher M West, Kirk D C Jensen. mBio. 2024 Sep 20:e0052724. doi: 10.1128/mbio.00527-24

Oxygen-dependent regulation of F-box proteins in Toxoplasma gondii is mediated by Skp1 glycosylation

Donna Huber on September 20, 2024

Figure 8. Immunolocalization of FBXO13-HA3 and FBXO14-HA3.

A dynamic proteome is required for cellular adaption to changing environments including levels of O2, and the SKP1/CULLIN-1/F-box protein/RBX1 (SCF) family of E3 ubiquitin ligases contributes importantly to proteasome-mediated degradation. We examine, in the apicomplexan parasite Toxoplasma gondii, the influence on the interactome of SKP1 by its novel glycan attached to a hydroxyproline generated by PHYa, the likely ortholog of the HIFα PHD2 oxygen-sensor of human host cells. Strikingly, the representation of several putative F-box proteins (FBPs) is substantially reduced in PHYaΔ parasites grown in fibroblasts. One, FBXO13, is a predicted lysyl hydroxylase related to the human JmjD6 oncogene except for its F-box domain. The abundance of FBXO13, epitope-tagged at its genetic locus, was reduced in PHYaΔ parasites thus explaining its diminished presence in the SKP1 interactome. A similar effect was observed for FBXO14, a cytoplasmic protein of unknown function that may have co-evolved with PHYa in apicomplexans. Similar findings in glycosylation-mutant cells, rescue by proteasomal inhibitors, and unchanged transcript levels, suggested the involvement of the SCF in their degradation. The effect was selective, because FBXO1 was not affected by loss of PHYa. These findings are physiologically significant because the effects were phenocopied in parasites reared at 0.5% O2. Modest impact on steady-state SKP1 modification levels suggests that effects are mediated during a lag phase in hydroxylation of nascent SKP1. The dependence of FBP abundance on O2-dependent SKP1 modification likely contributes to the reduced virulence of PHYaΔ parasites owing to impaired ability to sense O2 as an environmental signal.

Msano N Mandalasi, Elisabet Gas-Pascual, Carlos Gustavo Baptista, Bowen Deng, Hanke van der Wel, John A W Kruijtzer, Geert-Jan Boons, Ira J Blader, Christopher M West. J Biol Chem. 2024 Sep 20:107801. doi: 10.1016/j.jbc.2024.107801.

A combination of four Toxoplasma gondii nuclear-targeted effectors protects against interferon gamma-driven human host cell death

Donna Huber on September 18, 2024

Fig 1 IFNγ stimulation following infection is countered by MYR1, preventing early tachyzoite egress and host cell death.

In both mice and humans, Type II interferon gamma (IFNγ) is crucial for the regulation of Toxoplasma gondii (T. gondii) infection, during acute or chronic phases. To thwart this defense, T. gondii secretes protein effectors hindering the host’s immune response. For example, T. gondii relies on the MYR translocon complex to deploy soluble dense granule effectors (GRAs) into the host cell cytosol or nucleus. Recent genome-wide loss-of-function screens in IFNγ-primed primary human fibroblasts identified MYR translocon components as crucial for parasite resistance against IFNγ-driven vacuole clearance. However, these screens did not pinpoint specific MYR-dependent GRA proteins responsible for IFNγ signaling blockade, suggesting potential functional redundancy. Our study reveals that T. gondii depends on the MYR translocon complex to prevent parasite premature egress and host cell death in human cells stimulated with IFNγ post-infection, a unique phenotype observed in various human cell lines but not in murine cells. Intriguingly, inhibiting parasite egress did not prevent host cell death, indicating this mechanism is distinct from those described previously. Genome-wide loss-of-function screens uncovered TgIST, GRA16, GRA24, and GRA28 as effectors necessary for a complete block of IFNγ response. GRA24 and GRA28 directly influenced IFNγ-driven transcription, GRA24’s action depended on its interaction with p38 MAPK, while GRA28 disrupted histone acetyltransferase activity of CBP/p300. Given the intricate nature of the immune response to T. gondii, it appears that the parasite has evolved equally elaborate mechanisms to subvert IFNγ signaling, extending beyond direct interference with the JAK/STAT1 pathway, to encompass other signaling pathways as well.

Henry B, Phillips AJ, Sibley LD, Rosenberg A. 2024. mBio 0:e02124-24. https://doi.org/10.1128/mbio.02124-24

Tag: Toxoplasma gondii