bims-toxgon 2026-04-19 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2026–04–19
nine papers selected by
Lakesh Kumar, BITS Pilani

Synergy between upregulated small GTPase immunity-associated proteins and lysosomal ATP6V1D in restricting intracellular Toxoplasma growth.
Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system.
Molecular features of myosin F adapted for driving actin flows in Toxoplasma gondii.
Dynamic landscape of microRNA expression in the feline small intestine during Toxoplasma gondii infection.
Toxoplasma gondii infection induces extracellular vesicle miRNAs in synaptic plasticity and neural mechanisms.
Screening of purine nucleoside analogues against intracellular Toxoplasma gondii.
Cellular Target Engagement and Dissociation Kinetics of Class I-Selective Histone Deacetylase (HDAC) Inhibitors.
GID/CTLH E3 ligase complex control cell fate programs for sexual development of Plasmodium falciparum.
Genetically encoded manipulation of ATP/ADP ratio in human cells uncovers proteomic and physiological signatures of energy stress.

Microbiol Spectr. 2026 Apr 13. e0394725

Synergy between upregulated small GTPase immunity-associated proteins and lysosomal ATP6V1D in restricting intracellular Toxoplasma growth.

Md Mukthar Mia, Shahbaz M Khan, Abdur Rehman Azam, Jordan Reilly, William H Witola.

  Toxoplasma gondii is an obligate intracellular zoonotic protozoan parasite that can cause severe neonatal disease in congenitally acquired infections and severe opportunistic illnesses in immunocompromised individuals. Intracellularly, T. gondii evades host cell defense mechanisms by residing within a specialized non-fusogenic membranous compartment known as the parasitophorous vacuole (PV). Notwithstanding this, we have previously shown that upregulation of small GTPase immunity-associated proteins (GIMAPs) during T. gondii infection promotes fusion of lysosomes with the PV membrane (PVM), resulting in restriction of parasite replication in the T. gondii-refractory Lewis (LEW) rat. Herein, we found that, in addition to GIMAPs, the LEW rat upregulates ATP6V1D, a subunit of the lysosomal vacuolar-type H+-ATPase (v-ATPase) proton pump, in response to T. gondii infection. Disruption of ATP6V1D by CRISPR/Cas9 in a rat macrophage (NR8383) cell line impaired v-ATPase function, leading to defective lysosomal acidification and a concomitant increase in intracellular T. gondii growth. Intriguingly, overexpression of GIMAP 5 or 6 transgene in the ∆ATP6V1D mutant cell line reduced parasite growth by 2.5- and 5-fold, respectively. Comparatively, overexpression of GIMAP 5 or 6 transgene in the wild-type NR8383 cell line had at least threefold more robust effect of reducing parasite growth than in the ∆ATP6V1D mutant cell. Together, our findings imply that while GIMAP-induced activation of lysosome translocation to the PVM is important for restricting parasite growth, concomitant upregulation of the ATP6V1D plays an additively significant role through enhanced acidification of lysosomes to activate hydrolytic enzymes that degrade the parasites following PVM fusion.IMPORTANCEToxoplasma gondii is a highly prevalent parasite that causes severe illnesses in congenitally infected neonates and in immunocompromised individuals. No completely effective drugs nor vaccines exist against T. gondii. Thus, understanding innate mechanisms that resistant hosts employ to orchestrate defenses against the parasite could unveil new strategies for control of toxoplasmosis. Herein, we analyzed transcriptomic data of the T. gondii-resistant rat (Lewis) and identified small GTPase immunity-associated proteins (GIMAPs), and lysosome-associated proteins (including ATP6V1D) that are upregulated in response to infection. We investigated the interplay between those proteins and found that upregulated expression of GIMAPs drives lysosomes to fuse with the parasite vacuole, while upregulated ATP6V1D facilitates lysosomal acidification, which is important for activation of degradative lysosomal enzymes. Those events culminated in the restriction of intracellular T. gondii growth. Collectively, our findings indicate that upregulation of GIMAPs and ATP6V1D orchestrates synergistic mechanisms that contribute to inhibition of intracellular T. gondii growth.

Keywords:  ATP6V1D; GIMAP; Lewis rat; Toxoplasma inhibition; lysosome

DOI:  https://doi.org/10.1128/spectrum.03947-25
Parasit Vectors. 2026 Apr 13.

Functional characterization of 11 novel rhoptry proteins in the type I RH strain of Toxoplasma gondii using the CRISPR-Cas9 system.

Hong-Yu Song, Hui Cao, Shi-Bo Huang, Hany M Elsheikha, Zhi Zheng, Xin-Sheng Lu, Xing Tian, Xiao-Nan Zheng, Xing-Quan Zhu.

   BACKGROUND: Rhoptry proteins (ROPs) are secreted effectors that play important roles in the virulence of Toxoplasma gondii by facilitating host cell invasion and immune modulation. Although many ROPs have been predicted, their specific functions remain largely unexplored. This study investigates the roles of 11 previously uncharacterized ROPs in T. gondii biology, with a focus on their contributions to virulence.
METHODS: Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing was employed to generate epitope-tagged and knockout mutants for each candidate ROP in the T. gondii RHΔku80 strain. Subcellular localization was determined via immunofluorescence microscopy in both tachyzoite and bradyzoite stages. In vitro assays assessed parasite invasion, replication, egress, and plaque formation. In vivo virulence was evaluated in mouse infection models. To explore molecular mechanisms underlying virulence attenuation, we performed transcriptomic profiling of RHΔrop64 and RHΔrop65 knockout strains.
RESULTS: All 11 candidate ROPs exhibited rhoptry localization in both tachyzoite and bradyzoite stages. Despite no apparent in vitro growth defects, deletion of ROP64 and ROP65 led to significant attenuation of virulence in mice, with ROP64 showing the most pronounced effect. Transcriptome analysis revealed downregulation of key immune-modulatory genes, including ROP5, ROP39, TgIST, and PLP1. In addition, RHΔrop64 exhibited broader suppression of ROPs than RHΔrop65, suggesting it has a more pronounced role in immune modulation.
CONCLUSIONS: ROP64 and ROP65 are critical to T. gondii virulence, likely through modulation of the parasite's immune-evasive machinery. Their regulatory influence on effector expression underscores their importance in host adaptation. Importantly, the RHΔrop64 mutant displays characteristics of an attenuated strain with potential for vaccine development against toxoplasmosis.

Keywords:   Toxoplasma gondii ; Clustered regularly interspaced short palindromic repeats; Clustered regularly interspaced short palindromic repeats-associated protein 9; Rhoptry proteins; Transcriptomics; Virulence

DOI:  https://doi.org/10.1186/s13071-026-07387-0
J Cell Sci. 2026 Apr 01. pii: jcs264520. [Epub ahead of print]139(7):

Molecular features of myosin F adapted for driving actin flows in Toxoplasma gondii.

Thomas E Sladewski, Aoife T Heaslip.

  Toxoplasma gondii is a single-celled apicomplexan parasite that relies on a highly polarized endomembrane system for its invasion of, and survival within, host cells. Recent advances in imaging technologies have revealed that vesicle transport and the organization of organelles within the endomembrane pathway require a highly dynamic actin cytoskeleton. These dynamics in turn rely on the activity of myosin F (MyoF), a molecular motor unique to alveolates. The defining characteristic of this molecular motor is a WD40 β-propeller domain, exclusively found in this class of myosin. To understand the mechanism by which MyoF controls the dynamics and organization of actin, we studied the biophysical properties of the purified motor in vitro. A MyoF construct lacking its WD40 tail domain (MyoF-Motor) is dimeric and can bind and translocate actin in an in vitro motility assay. Single-molecule studies show that the dimeric construct is non-processive; however, small ensembles move inefficiently on single filaments of skeletal actin. In contrast, single molecules of the full-length motor move processively on Toxoplasma actin and jasplakinolide-stabilized skeletal actin bundles. Electron microscopy of negatively stained images of MyoF and quantitative size exclusion chromatography show that the WD40 domain oligomerizes to form a complex containing multiple dimeric molecules, which provides an explanation for why the full-length motor is processive compared to the dimeric MyoF-Motor construct. Finally, we show that MyoF binds microtubules through its WD40 domain and can slide actin filaments relative to microtubules. We propose a model whereby MyoF oligomers drive actin dynamics by translocating filaments relative to the cytoskeleton of the parasite. These molecular features provide new insight into how MyoF functions in the cell to regulate actin organization during vesicle transport.

Keywords:   Toxoplasma gondii ; Actin; Microtubule; Myosin

DOI:  https://doi.org/10.1242/jcs.264520
Parasit Vectors. 2026 Apr 11.

Dynamic landscape of microRNA expression in the feline small intestine during Toxoplasma gondii infection.

Bintao Zhai, Bibo Bao, Shi-Chen Xie, Hui Yang, Yang Liu, Weiwei Wang, Yaxin Zhou, Bing Li, Junjun He, Jiyu Zhang.

   BACKGROUND: Toxoplasma gondii, an obligate intracellular parasite with felids as its definitive hosts, undergoes sexual reproduction and oocyst shedding in the feline small intestine, a critical stage for its transmission. Small non-coding RNAs, particularly microRNAs (miRNAs), are crucial post-transcriptional regulators in host-pathogen interactions, but their role in the definitive host's intestine during T. gondii infection remains unexplored.
METHODS: Fifteen cats were divided into control, primary infection (6, 10, 14 days post-infection, DPI), and secondary infection (SI) groups. Infection was confirmed via B1 gene polymerase chain reaction (PCR). Small RNA sequencing was performed on the ileal epithelium. Bioinformatics analyses identified known and novel miRNAs, differential expression, target genes, and enriched pathways. Key miRNA-messenger RNA (mRNA) interactions were validated by dual-luciferase assay, and sequencing results were confirmed by quantitative PCR (qPCR).
RESULTS: Successful infection was molecularly confirmed. Sequencing identified 2666 miRNAs (2575 known, 91 novel). A dynamic pattern of differentially expressed (DE) miRNAs was observed, with peaks at 6 DPI (126), 10 DPI (122), 14DPI (36) and SI DPI (237), coinciding with active oocyst shedding. Key miRNAs like hsa-miR-199b-5p and ssc-miR-199b-5p were persistently downregulated. Target prediction and network analysis revealed complex interactions, including miR-199b-5p targeting CYTH1 and COQ7. Functional enrichment highlighted significant involvement of target genes in the Rap1 and AMPK signaling pathways, as well as processes related to development and cellular organization. The novel_538-CNN2 interaction was experimentally validated.
CONCLUSIONS: This study provides the first comprehensive profile of miRNA expression in the feline small intestine during T. gondii infection. The temporal dynamics and specific dysregulation of miRNAs, coupled with enrichment in key pathways controlling cell adhesion and metabolism, suggest that T. gondii could orchestrate a sophisticated post-transcriptional program in its definitive host to potentially modify the intestinal environment for successful oocyst production and shedding. These findings lay the groundwork for future functional studies regarding the interplay between T. gondii and its definitive hosts.

Keywords:   Toxoplasma gondii ; Feline; Host-pathogen interaction; MicroRNA; Small intestine

DOI:  https://doi.org/10.1186/s13071-026-07356-7
Extracell Vesicles Circ Nucl Acids. 2026 ;7(1): 1-15

Toxoplasma gondii infection induces extracellular vesicle miRNAs in synaptic plasticity and neural mechanisms.

Poppy Cairney, Elton Rosas de Vasconcelos, Glenn A McConkey.

  Aim: This study investigates the change in profiles of miRNAs in extracellular vesicles released during Toxoplasma gondii (T. gondii) infection. T. gondii has been implicated in host behavioural modifications and neuroinflammatory responses, yet the molecular mechanisms involved in these changes remain poorly understood. Extracellular vesicles, involved in intercellular communication, play an important role in host-pathogen interactions, particularly through the transfer of microRNAs (miRNAs); however, the impact of extracellular vesicle miRNAs in T. gondii infection remains largely unexplored. Methods: Human BE(2)-M17 neuronal cells were infected with Toxoplasma gondii to investigate infection-induced changes in extracellular vesicle (EV) miRNA content. EVs from infected and control cultures were isolated, characterised, and subjected to miRNA extraction followed by next-generation sequencing and differential expression analysis using standard bioinformatic pipelines. Predicted miRNA targets were integrated across multiple databases and analysed for enriched pathways to identify neuronal regulatory networks. Results: Pathway network analysis identified key neurobiological pathways, including neuroplasticity, neurotransmission, and neuroinflammation in high-confidence miRNA targets with gene enrichment of neurotrophin and long-term depression and long-term potentiation, which may underlie parasite-induced alterations in neural function. Bioinformatic analysis of extracellular vesicle miRNA profiles from infected and uninfected neuronal cells revealed a set of miRNAs including hsa-miR-4645-3p with significant upregulation in response to infection. Conclusion: These findings suggest that T. gondii modulates host neuronal processes through extracellular vesicle-mediated miRNA transfer, providing a potential mechanistic link between infection and parasite-associated cognitive and neuropsychiatric disturbances.

Keywords:  Pathogen; brain; dopamine; exosome; infectious disease; neuronal; neurotransmission; noncoding RNA

DOI:  https://doi.org/10.20517/evcna.2025.34
Int J Parasitol Drugs Drug Resist. 2026 Apr 10. pii: S2211-3207(26)00011-4. [Epub ahead of print]31 100641

Screening of purine nucleoside analogues against intracellular Toxoplasma gondii.

Hamza A A Elati, Serge Van Calenbergh, Lilach Sheiner, Harry P de Koning.

  Toxoplasmosis remains a world-wide public health concern, especially for the immunocompromised. Although this population segment is increasing due to therapeutic interventions, organ transplants and infections including HIV, treatment relies almost exclusively on sulfadoxine and pyrimethamine, antifolates developed against malaria but with only moderate efficacy against acute toxoplasmosis and no effect on the chronic stage. Here we explore whether 7-substituted analogues of 7-deazaadenosine (tubercidin) that have shown remarkable efficacy against other protozoan pathogens, might also show anti-toxoplasmic activity. Tubercidin and a series of eleven 7-substituted analogues including 2'-deoxy and 3'-deoxyribofuranoses was tested against intracellular Toxoplasma gondii tachyzoites. The test compounds yielded EC50 values between 0.012 and 1.72 μM, well below those of the control drug sulfadiazine (11.9 μM) and the previously identified purine analogue adenosine arabinoside (Ara-A; 11.4 μM). The tubercidin analogues displayed at most moderate toxicity to HFF cells, with the most efficacious compound, 7-(3,4-di-Cl-phenyl)-3'-deoxytubercidin (FH8513) reaching a selectivity index of >2500. These nucleosides are most likely taken up by T. gondii through one of the four Equilibrative Nucleoside Transporters (ENTs) encoded by the parasites. However, deletion of TgENT2 and/or TgENT3 had no effect on the EC50 values, and deletion of TgAT1 actually sensitised the tachyzoites to most of the tubercidin analogues. We propose that these nucleosides are internalised through the TgENT1 uridine transporter and that the sensitisation in ΔTgAT1 cells is the result of reduced uptake of adenosine that competes with the tubercidin analogues for metabolic enzymes such as adenosine kinase.

Keywords:  Chemotherapy; Nucleoside transporter; Purine antimetabolite; Toxoplasmosis; Tubercidin

DOI:  https://doi.org/10.1016/j.ijpddr.2026.100641
Int J Mol Sci. 2026 Mar 26. pii: 3036. [Epub ahead of print]27(7):

Cellular Target Engagement and Dissociation Kinetics of Class I-Selective Histone Deacetylase (HDAC) Inhibitors.

Irina Honin, Zora Novakova, Felix Feller, Simon Schneider, Linda Schäker-Hübner, Cyril Barinka, Finn K Hansen.

  Histone deacetylases (HDACs) 1-3 are key regulators of gene expression and represent important therapeutic targets in cancer, neurodegenerative, and immune disorders. Many potent class I HDAC inhibitors display slow- and tight-binding kinetics, which profoundly influence their efficacy and pharmacodynamics. In particular, their dissociation rate (off-kinetic) is critical, since prolonged target engagement greatly influences drug efficacy in vivo. However, the off-kinetics of HDAC inhibitors are often overlooked in the early stages of drug development. Here, we investigated the dissociation kinetics of tucidinostat, trapoxin A, and TNG260 in comparison to the pan-HDAC inhibitor vorinostat. Using biochemical 100-fold jump dilution assays, NanoBRET assays, and cellular washout experiments, we characterized the dissociation of these compounds from purified proteins and in a cellular context. Tucidinostat showed moderately slow off-kinetics, while the clinical candidate TNG260 demonstrated pronounced tight-binding properties. Trapoxin A displayed remarkable discrepancies between assays, as it showed fast dissociation kinetics in the biochemical assay, but tight-binding properties in a cellular setting. These findings not only address the previously unexplored dissociation kinetics of two clinically relevant inhibitors, but also underscore the importance of comprehensive kinetic profiling of novel HDAC inhibitors in cellular models.

Keywords:  HDAC inhibitor (HDACi); NanoBRET; NanoBiT; dissociation behavior; epigenetics; histone deacetylase (HDAC); residence time

DOI:  https://doi.org/10.3390/ijms27073036
Nat Commun. 2026 Apr 15. pii: 3497. [Epub ahead of print]17(1):

GID/CTLH E3 ligase complex control cell fate programs for sexual development of Plasmodium falciparum.

Danushka S Marapana, Sash Lopaticki, Balu Balan, Kitsanapong Reaksudsan, Simon A Cobbold, Niall D Geoghegan, Sachin Khurana, Stephen W Scally, Peter Hickey, Niva Jayakrishnan, Vineet Vaibhav, Sukhdeep Spall, Jumana M Yousef, Laura F Dagley, Christopher D Goodman, Anton Cozijnsen, Geoffrey I McFadden, Kelly L Rogers, Aaron Jex, David Komander, James S McCarthy, Christopher J Tonkin, Matthew W A Dixon, Alan F Cowman.

Transmission of the malaria parasite Plasmodium falciparum requires the formation of specialised sexual cells called gametocytes. A hallmark of P. falciparum gametocyte development is its long duration, during which the parasite undergoes dramatic cellular remodelling including morphological, physiological and metabolic changes which result in the formation of a transmission ready, stage V gametocyte. Here we show that the PfGID E3 ubiquitin ligase complex regulates critical gametocyte cell fate programmes through the targeted ubiquitination of key proteins. Deletion of PfGID complex components leads to an arrest in gametocyte development and a loss of transmission to mosquitoes. PfGID governs gametocyte development by fine-tuning the protein levels of two substrates: the ZFP36 family RNAbinding protein GD1, and PfDPL, a cryptochrome-like protein. Our findings reveal that PfDPL regulates the expression of male-specific proteins early in gametocyte development that are essential for gametogenesis. In parallel to the PfDPL controlled cell fate program the RNA binding protein GD1 regulates transcripts crucial for gametocyte development by holding them in a state of translational repression. These findings illuminate the intricate molecular choreography underlying Plasmodium sexual development and provide insights into how single-celled eukaryotes execute cell-fate programmes to navigate complex life cycles and adapt to diverse host environments.

DOI: https://doi.org/10.1038/s41467-026-69183-9
Cell Chem Biol. 2026 Apr 13. pii: S2451-9456(26)00074-7. [Epub ahead of print]

Genetically encoded manipulation of ATP/ADP ratio in human cells uncovers proteomic and physiological signatures of energy stress.

Alex E Ekvik, Megan M Kober, Denis V Titov.

  The ability of cells to power energy-demanding processes depends on maintaining the ATP hydrolysis reaction a billion-fold away from equilibrium. Cells respond to alterations in the energy state by sensing changes in the ratio of ATP, ADP, AMP, and inorganic phosphate levels. A key barrier to understanding how this happens is a lack of tools for direct manipulation of the energy state in living cells. Here, we introduce ATPGobble-a genetically encoded tool that hydrolyzes ATP in vivo. ATPGobble increases the metabolic rate, decreases [ATP]/[ADP] and [ATP]/[AMP] ratios, and activates AMPK in human cells. We performed a systematic analysis of proteome and phosphoproteome changes caused by ATPGobble, and found that it remodels cytoskeleton, cell cycle, and translation machinery. Our results establish ATPGobble as a powerful new tool for dissecting the regulatory roles of energy state in living cells.

Keywords:  AMPK; ATP; ATP/ADP ratio; F1 ATPase; cell fitness; energy status; energy stress; genetically encoded tools; phosphoproteomics; proteomics

DOI:  https://doi.org/10.1016/j.chembiol.2026.03.004