bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2024–12–15
seven papers selected by
Lakesh Kumar, BITS Pilani
  1. Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants.
  2. Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.
  3. MULTIPLE, REDUNDANT CARBOXYLIC ACID TRANSPORTERS SUPPORT MITOCHONDRIAL METABOLISM IN PLASMODIUM FALCIPARUM.
  4. Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis.
  5. A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.
  6. Emergent actin flows explain distinct modes of gliding motility.
  7. Characterization of molecular interactions between HDAC7 and MEF2A.
  1. bioRxiv. 2024 Nov 27. pii: 2024.11.27.625683. [Epub ahead of print]
    Metabolic Adaptability and Nutrient Scavenging in Toxoplasma gondii: Insights from Ingestion Pathway-Deficient Mutants.
    Patrick A Rimple, Einar B Olafsson, Benedikt M Markus, Fengrong Wang, Leonardo Augusto, Sebastian Lourido, Vern B Carruthers.
      The obligate intracellular parasite Toxoplasma gondii replicates within a specialized compartment called the parasitophorous vacuole (PV). Recent work showed that despite living within a PV, Toxoplasma endocytoses proteins from the cytosol of infected host cells via a so-called ingestion pathway. The ingestion pathway is initiated by dense granule protein GRA14, which binds host ESCRT machinery to bud vesicles into the lumen of the PV. The protein-containing vesicles are internalized by the parasite and trafficked to the Plant Vacuole-like compartment (PLVAC), where cathepsin protease L (CPL) degrades the cargo and the chloroquine resistance transporter (CRT) exports the resulting peptides and amino acids to the parasite cytosol. However, although the ingestion pathway was proposed to be a conduit for nutrients, there is limited evidence for this hypothesis. We reasoned that if Toxoplasma uses the ingestion pathway to acquire nutrients, then parasites lacking GRA14, CPL, or CRT should rely more on biosynthetic pathways or alternative scavenging pathways. To explore this, we conducted a genome-wide CRISPR screen in wild-type (WT) parasites and Δ gra14 , Δ cpl , and Δ crt mutants to identify genes that become more fitness conferring in ingestion-deficient parasites. Our screen revealed a significant overlap of genes that become more fitness conferring in the ingestion mutants compared to WT. Pathway analysis indicated that Δ cpl and Δ crt mutants relied more on pyrimidine biosynthesis, fatty acid biosynthesis, TCA cycle, and lysine degradation. Bulk metabolomic analysis showed reduced levels of glycolytic intermediates and amino acids in the ingestion mutants compared to WT, highlighting the pathway's potential role in host resource scavenging. Interestingly, ingestion mutants showed an exacerbated growth defect when grown in amino acid-depleted media, suggesting a role for the Toxoplasma ingestion pathway during nutrient scarcity.
    Importance: Toxoplasma gondii is an obligate intracellular pathogen that infects virtually any nucleated cell in most warm-blooded animals. Infections are asymptomatic in most cases but people with weakened immunity can experience severe disease. For the parasite to replicate within the host, it must efficiently acquire essential nutrients, especially as it is unable to make several key metabolites. Understanding the mechanisms by which Toxoplasma scavenges nutrients from the host is crucial for identifying potential therapeutic targets. Our study highlights the function of the ingestion pathway in sustaining parasite metabolites and contributes to parasite replication under amino acid limiting conditions. This work advances our understanding of the metabolic adaptability of Toxoplasma .
    DOI:  https://doi.org/10.1101/2024.11.27.625683
  2. Elife. 2024 Dec 10. pii: RP102592. [Epub ahead of print]13
    Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.
    Francesca Torelli, Diogo M da Fonseca, Simon W Butterworth, Joanna C Young, Moritz Treeck.
      Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.
    Keywords:  Toxoplasma; crispr screening; immunology; infection; infectious disease; microbiology; parasitology
    DOI:  https://doi.org/10.7554/eLife.102592
  3. bioRxiv. 2024 Nov 27. pii: 2024.11.26.624872. [Epub ahead of print]
    MULTIPLE, REDUNDANT CARBOXYLIC ACID TRANSPORTERS SUPPORT MITOCHONDRIAL METABOLISM IN PLASMODIUM FALCIPARUM.
    Krithika Rajaram, Gabriel W Rangel, Justin T Munro, Sethu C Nair, Manuel Llinás, Sean T Prigge.
      The mitochondrion of the deadliest human malaria parasite, Plasmodium falciparum, is an essential source of cellular acetyl-CoA during the asexual blood-stage of the parasite life cycle. Blocking mitochondrial acetyl-CoA synthesis leads to a hypoacetylated proteome and parasite death. We previously determined that mitochondrial acetyl-CoA is primarily synthesized from glucose-derived pyruvate by α-ketoacid dehydrogenases. Here, we asked if inhibiting the import of glycolytic pyruvate across the mitochondrial inner membrane would affect acetyl-CoA production and, thus, could be a potential target for antimalarial drug development. We selected the two predicted mitochondrial pyruvate carrier proteins ( Pf MPC1 and Pf MPC2) for genetic knockout and isotopic metabolite tracing via HPLC-MS metabolomic analysis. Surprisingly, we observed that asexual blood-stage parasites could survive the loss of either or both Pf MPCs with only minor growth defects, despite a substantial reduction in the amount of glucose-derived isotopic labelling into acetyl-CoA. Furthermore, genetic deletion of two additional mitochondrial carboxylic acid transporters - DTC (di/tricarboxylic acid carrier) and YHM2 (a putative citrate/α-ketoglutarate carrier protein) - only mildly affected asexual blood-stage replication, even in the context of Pf MPC deficiency. Although we observed no added impact on the incorporation of glucose carbon into acetyl-CoA in these quadruple knockout mutants, we noted a large decrease in glutamine-derived label in tricarboxylic acid cycle metabolites, suggesting that DTC and YHM2 both import glutamine derivatives into the mitochondrion. Altogether, our results expose redundant routes used to fuel the blood-stage malaria parasite mitochondrion with imported carbon from two major sources - glucose and glutamine.
    SIGNIFICANCE: The mitochondrion of malaria parasites generates key molecules, such as acetyl-CoA, that are required for numerous cellular processes. To support mitochondrial biosynthetic pathways, the parasites must transport carbon sources into this organelle. By studying how the mitochondrion obtains pyruvate, a molecule derived from glucose, we have uncovered redundant carbon transport systems that ensure parasite survival in red blood cells. This metabolic redundancy poses a challenge for drug development, as it enables the parasite to adapt and survive by relying on alternative pathways when one is disrupted.
    DOI:  https://doi.org/10.1101/2024.11.26.624872
  4. Elife. 2024 Dec 11. pii: RP100256. [Epub ahead of print]13
    Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis.
    Amanda Mixon Blackwell, Yasaman Jami-Alahmadi, Armiyaw S Nasamu, Shota Kudo, Akinobu Senoo, Celine Slam, Kouhei Tsumoto, James A Wohlschlegel, Jose Manuel Martinez Caaveiro, Daniel E Goldberg, Paul A Sigala.
      Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 Å-resolution X-ray structure that revealed a highly α-helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.
    Keywords:  P. falciparum; RNA; apicoplast; biochemistry; chemical biology; gene expression; heme oxygenase; infectious disease; malaria; microbiology
    DOI:  https://doi.org/10.7554/eLife.100256
  5. Infect Dis Poverty. 2024 Dec 10. 13(1): 94
    A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.
    Yao Liang, Shi-Chen Xie, Yi-Han Lv, Yuan-Hui He, Xiao-Nan Zheng, Wei Cong, Hany M Elsheikha, Xing-Quan Zhu.
       BACKGROUND: Toxoplasma gondii oocysts, excreted in cat feces, pose a significant health risk to humans through contaminated soil and water. Rapid and accurate detection of T. gondii in environmental samples is essential for public health protection.
    METHODS: We developed a novel, single-tube detection method that integrates loop-mediated isothermal amplification (LAMP), the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system, and lateral flow immunoassay strips for rapid, visual identification of T. gondii. This method targets the T. gondii B1 gene, initially amplifies it with LAMP, directed by a single-guide RNA (sgRNA). It then recognizes the amplified target gene and activates trans-cleavage, cutting nearby single-stranded DNA (ssDNA) reporters. Fluorescence detection was performed using a 6-Carboxyfluorescein (FAM)-12N-Black Hole Quencher-1 (BHQ1) reporter, while Fluorescein Isothiocyanate (FITC)-12N-Biotin enabled visual detection on lateral flow strips. The method was tested for its ability to detect various T. gondii genotypes and related parasites, assessing its specificity and broad-spectrum applicability. It was further applied to real-world environmental samples to evaluate its practicality.
    RESULTS: The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability, successfully identifying nine T. gondii genotypes and distinguishing them from 11 other parasitic species. Sensitivity testing at both molecular (plasmid) and practical (oocyst) levels showed detection limits of 10  copies/μL and 0.1 oocyst, respectively. When applied to 112 environmental samples (soil, water, and cat feces), the method demonstrated 100% sensitivity, accurately reflecting known infection rates.
    CONCLUSIONS: This LAMP-CRISPR/Cas12b single-tube method offers a robust, innovative approach for monitoring zoonotic T. gondii in environmental samples, with significant implications for public health surveillance.
    Keywords:   Toxoplasma gondii ; CRISPR/Cas12b; Environmental detection; Lateral flow immunoassay; Loop-mediated isothermal amplification; Molecular diagnostics; Zoonotic parasites
    DOI:  https://doi.org/10.1186/s40249-024-01266-5
  6. Nat Phys. 2024 ;20(12): 1989-1996
    Emergent actin flows explain distinct modes of gliding motility.
    Christina L Hueschen, Li-Av Segev-Zarko, Jian-Hua Chen, Mark A LeGros, Carolyn A Larabell, John C Boothroyd, Rob Phillips, Alexander R Dunn.
      During host infection, Toxoplasma gondii and related unicellular parasites move using gliding, which differs fundamentally from other known mechanisms of eukaryotic cell motility. Gliding is thought to be powered by a thin layer of flowing filamentous (F)-actin sandwiched between the plasma membrane and a myosin-covered inner membrane complex. How this surface actin layer drives the various gliding modes observed in experiments-helical, circular, twirling and patch, pendulum or rolling-is unclear. Here we suggest that F-actin flows arise through self-organization and develop a continuum model of emergent F-actin flow within the confines provided by Toxoplasma geometry. In the presence of F-actin turnover, our model predicts the emergence of a steady-state mode in which actin transport is largely directed rearward. Removing F-actin turnover leads to actin patches that recirculate up and down the cell, which we observe experimentally for drug-stabilized actin bundles in live Toxoplasma gondii parasites. These distinct self-organized actin states can account for observed gliding modes, illustrating how different forms of gliding motility can emerge as an intrinsic consequence of the self-organizing properties of F-actin flow in a confined geometry.
    Keywords:  Biological physics; Cellular motility
    DOI:  https://doi.org/10.1038/s41567-024-02652-4
  7. J Biomol Struct Dyn. 2024 Dec 11. 1-10
    Characterization of molecular interactions between HDAC7 and MEF2A.
    Narayan Gautam, Prem P Chapagain, Narayan P Adhikari, Purushottam B Tiwari.
      Interactions of transcriptional corepressors such as histone deacetylase 7 (HDAC7), a class IIa HDAC, with myocyte enhancer factor-2 (MEF2) regulate MEF2 activity. Despite previous investigations exploring interactions between HDAC7 and MEF2, a detailed characterization of the HDAC7-MEF2 functional complex is still lacking. Herein, we first modeled the structure of the HDAC7-MEF2A complex and investigated the inter-protein interactions using all-atom molecular dynamics (MD) simulations. We identified specific amino acids within HDAC7 and MEF2A that participate in interactions such as salt bridges, hydrogen bonds, and hydrophobic interactions. Our results reveal a salt bridge formed between LYS96(HDAC7) and ASP63(MEF2A). Our analysis also predicted formations of reliable hydrogen bonds between SER82(HDAC7) and ASP63(MEF2A) as well as LYS96(HDAC7) and ASP63(MEF2A). In addition, clustering of hydrophobic residues at the interface contributes in stabilizing the HDAC7-MEF2A complex. Results from multiple sequence alignment show that most of the HDAC7 residues that are predicted to associate with MEF2A are conserved in at least three class IIa HDACs and all predicted residues in MEF2A are conserved in MEF2s. We also found that the association of DNA to MEF2A has no significant effect on HDAC7-MEF2A interactions. Our results may also provide useful insights into the interactions between other class IIa HDACs and MEF2s.
    Keywords:  HDAC7; MEF2A; hydrogen bonding; hydrophobic interactions; molecular dynamics simulations; salt bridge; solvent-accessible surface area
    DOI:  https://doi.org/10.1080/07391102.2024.2437523
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