bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2023–09–17
nine papers selected by
Lakesh Kumar, BITS Pilani



  1. bioRxiv. 2023 Aug 30. pii: 2023.08.29.555340. [Epub ahead of print]
      The cytoskeletal protein actin plays a critical role in the pathogenicity of Toxoplasma gondii , mediating invasion and egress, cargo transport, and organelle inheritance. Advances in live cell imaging have revealed extensive filamentous actin networks in the Apicomplexan parasite, but there is conflicting data regarding the biochemical and biophysical properties of Toxoplasma actin. Here, we imaged the in vitro assembly of individual Toxoplasma actin filaments in real time, showing that native, unstabilized filaments grow tens of microns in length. Unlike skeletal muscle actin, Toxoplasma filaments intrinsically undergo rapid treadmilling due to a high critical concentration, fast monomer dissociation, and rapid nucleotide exchange. Cryo-EM structures of stabilized and unstabilized filaments show an architecture like skeletal actin, with differences in assembly contacts in the D-loop that explain the dynamic nature of the filament, likely a conserved feature of Apicomplexan actin. This work demonstrates that evolutionary changes at assembly interfaces can tune dynamic properties of actin filaments without disrupting their conserved structure.
    DOI:  https://doi.org/10.1101/2023.08.29.555340
  2. bioRxiv. 2023 Sep 01. pii: 2023.08.31.555807. [Epub ahead of print]
      Toxoplasma gondii is a protozoan parasite that infects a broad spectrum of hosts and can colonize many organs and cell types. The ability to reside within a wide range of different niches requires substantial adaptability to diverse microenvironments. Very little is known about how this parasite senses various milieus and adapts its metabolism to survive, replicate during the acute stage, and then differentiate to the chronic stage. Most eukaryotes, from yeast to mammals, rely on a nutrient sensing machinery involving the TORC complex as master regulator of cell growth and cell cycle progression. The lysosome functions as a signaling hub where TORC complex assembles and is activated by transceptors, which both sense and transport amino acids, including the arginine transceptor SLC38A9. While most of the TORC components are lost in T. gondii , indicating the evolution of a distinct nutrient sensing mechanism, the parasite's lysosomal plant-like vacuolar compartment (PLVAC) may still serve as a sensory platform for controlling parasite growth and differentiation. Using SLC38A9 to query the T. gondii proteome, we identified four putative amino acid transporters, termed TgAAT1-4, that structurally resemble the SLC38A9 arginine transceptor. Assessing their expression and sub-cellular localization, we found that one of them, TgAAT1, localized to the PLVAC and is necessary for normal parasite extracellular survival and bradyzoite differentiation. Moreover, we show that TgAAT1 is involved in the PLVAC efflux of arginine, an amino acid playing a key role in T. gondii differentiation, further supporting the hypothesis that TgAAT1 might play a role in nutrient sensing.
    IMPORTANCE: T. gondii is a highly successful parasite infecting a broad range of warm-blood organisms including about one third of all humans. Although Toxoplasma infections rarely result in symptomatic disease in individuals with a healthy immune system, the incredibly high number of persons infected along with the risk of severe infection in immunocompromised patients and the potential link of chronic infection to mental disorders make this infection a significant public health concern. As a result, there is a pressing need for new treatment approaches that are both effective and well-tolerated. The limitations in understanding how Toxoplasma gondii manages its metabolism to adapt to changing environments and triggers its transformation into bradyzoites have hindered the discovery of vulnerabilities in its metabolic pathways or nutrient acquisition mechanisms to identify new therapeutic targets. In this work, we have shown that the lysosome-like organelle PLVAC, acting through the putative arginine transporter TgAAT1, plays a pivotal role in regulating the parasite's extracellular survival and differentiation into bradyzoites.
    DOI:  https://doi.org/10.1101/2023.08.31.555807
  3. Curr Protoc. 2023 Sep;3(9): e871
      Toxoplasma gondii is an obligate intracellular protozoan parasite that commonly infects mammals and birds throughout the world. This protocol describes murine models of acute T. gondii infection, toxoplasmic encephalitis and toxoplasma retinochoroiditis. T. gondii infection in severe combined immunodeficient (SCID) mice, deficient in T and B cells, has allowed for the study of T cell-independent mechanisms of defense against intracellular organisms, as described here. The uracil auxotroph strain cps1-1 and temperature-sensitive mutant strains of T. gondii induce protection against challenge with virulent strains of the parasite. They have allowed studies of immunization and adoptive-transfer experiments. A protocol is provided for infection with these mutant strains. The EGS strain of T. gondii has the unique feature of spontaneously forming tissue cysts in cell culture. Dual fluorescent reporter stains of this strain have allowed the study of tachyzoite to bradyzoite transitions in vitro and in vivo. A protocol for in vitro and in vivo growth of this strain and tissue cyst isolation is provided. Genetic manipulation of T. gondii and mice has led to the development of parasites that express fluorescent proteins as well as mice with fluorescently labeled leukocytes. This together with the use of T. gondii that express model antigens and transgenic mice that express the appropriate T cell receptor have facilitated the in vivo study of parasite host-interaction. In addition, parasites that express bioluminescent markers have made it possible to study the dynamics of infection in real time using bioluminescence imaging. Support protocols present methodology for evaluation of progression of infection and immune response to the parasite that includes these newer methodologies. In addition, support protocols address the maintenance of T. gondii tissue cysts and tachyzoites, as well as preparation of T. gondii lysate antigens. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Induction of acute T. gondii infection in mice Basic Protocol 2: Model of toxoplasmic encephalitis and toxoplasma retinochoroiditis in chronically infected mice Basic Protocol 3: Assessment of T. gondii invasion into neural tissue Basic Protocol 4: T. gondii infection in scid/scid (SCID) mice Basic Protocol 5: Infection with the uracil auxotroph strain CPS1-1 or the temperature-sensitive TS-4 strain of T. gondii Basic Protocol 6: In vivo and in vitro maintenance of the EGS strain of T. gondii Support Protocol 1: Assessment of progression of infection and immune response to T. gondii Support Protocol 2: Maintenance of a bank of T. gondii cysts of the ME49 strain Support Protocol 3: Maintenance of T. gondii tachyzoites using human foreskin fibroblasts Support Protocol 4: Maintenance of T. gondii tachyzoites in mice Support Protocol 5: Preparation of T. gondii lysate antigens Support Protocol 6: Isolation of T. gondii tissue cysts from brain.
    Keywords:  cellular immunity; encephalitis; humoral immunity; parasite; retinitis
    DOI:  https://doi.org/10.1002/cpz1.871
  4. J Biol Chem. 2023 Sep 11. pii: S0021-9258(23)02256-1. [Epub ahead of print] 105228
      The enzyme cofactor (R)-lipoic acid plays a critical role in central carbon metabolism due to its catalytic function in the generation of acetyl-CoA, which links glycolysis with the tricarboxylic acid cycle. This cofactor is also essential for the generation of succinyl CoA within the tricarboxylic acid cycle. However, the biological functions of (R)-lipoic acid extend beyond metabolism owing to its facile redox chemistry. Most recently, the reduced form of (R)-lipoic acid, (R)-dihydrolipoic acid, has been shown to inhibit histone deacetylases (HDACs) with selectivity for the inhibition of HDAC6. Here, we report the 2.4 Å-resolution X-ray crystal structure of the complex between (R)-dihydrolipoic acid and HDAC6 catalytic domain 2 from Danio rerio, and we report a dissociation constant (KD) of 350 nM for this complex as determined by isothermal titration calorimetry. The crystal structure illuminates key affinity determinants in the enzyme active site, including thiolate-Zn2+ coordination and S-π interactions in the F583-F643 aromatic crevice. This study provides the first visualization of the connection between HDAC function and the biological response to oxidative stress: the dithiol moiety of (R)-dihydrolipoic acid can serve as a redox-regulated pharmacophore capable of simultaneously targeting the catalytic Zn2+ ion and the aromatic crevice in the active site of HDAC6.
    Keywords:  HDAC; enzyme inhibitor; isothermal titration calorimetry; metalloenzyme; protein crystallography
    DOI:  https://doi.org/10.1016/j.jbc.2023.105228
  5. Proc Natl Acad Sci U S A. 2023 Sep 19. 120(38): e2302489120
      Loss of estrogen receptor (ER) pathway activity promotes breast cancer progression, yet how this occurs remains poorly understood. Here, we show that serine starvation, a metabolic stress often found in breast cancer, represses estrogen receptor alpha (ERα) signaling by reprogramming glucose metabolism and epigenetics. Using isotope tracing and time-resolved metabolomic analyses, we demonstrate that serine is required to maintain glucose flux through glycolysis and the TCA cycle to support acetyl-CoA generation for histone acetylation. Consequently, limiting serine depletes histone H3 lysine 27 acetylation (H3K27ac), particularly at the promoter region of ER pathway genes including the gene encoding ERα, ESR1. Mechanistically, serine starvation impairs acetyl-CoA-dependent gene expression by inhibiting the entry of glycolytic carbon into the TCA cycle and down-regulating the mitochondrial citrate exporter SLC25A1, a critical enzyme in the production of nucleocytosolic acetyl-CoA from glucose. Consistent with this model, total H3K27ac and ERα expression are suppressed by SLC25A1 inhibition and restored by acetate, an alternate source of acetyl-CoA, in serine-free conditions. We thus uncover an unexpected role for serine in sustaining ER signaling through the regulation of acetyl-CoA metabolism.
    Keywords:  SLC25A1; breast cancer; estrogen receptor; histone acetylation; serine metabolism
    DOI:  https://doi.org/10.1073/pnas.2302489120
  6. Infez Med. 2023 ;31(3): 407-410
      We report the case of a 58-year-old male with a recent diagnosis of HIV infection admitted for progressive muscular weakness and psychomotor impairment. Cerebrospinal examination documented a mild hyperproteinorrachia, with normal cells count and reduced glycorrhachia. Brain gadolinium-enhanced MRI showed bilateral T2 and FLAIR hyperintensities in the nucleo-capsular region and irregular contrast-enhancement of the globi pallidi and the right putamen. The histologic analysis of a quadriceps biopsy showed several foci of inflammatory infiltrates with concomitant muscular fiber atrophy and degeneration. Scattered intracytoplasmic inclusions were observed in muscle fibers, representing the main pathological feature. A positive PCR for Toxoplasma gondii and a Toxoplasma gondii specific monoclonal antibody immunohistochemical staining confirmed the diagnosis.
    Keywords:  HIV; Toxoplasma gondii; intracytoplasmic inclusion; muscle; myositis
    DOI:  https://doi.org/10.53854/liim-3103-16
  7. Eur J Med Chem. 2023 Sep 09. pii: S0223-5234(23)00767-5. [Epub ahead of print]261 115800
      Histone deacetylases (HDACs) catalyze the hydrolysis of acetyl-l-lysine side chains in histones and non-histones, which are key to epigenetic regulation in humans. Targeting HDACs has emerged as a promising strategy for treating various types of cancer, including myeloma and hematologic malignancies. At present, numerous small molecule inhibitors targeting HDACs are actively being investigated in clinical trials. Despite their potential efficacy in cancer treatment, HDAC inhibitors suffer from multi-directional selectivity and preclinical resistance issues. Hence, developing novel inhibitors based on cutting-edge medicinal chemistry techniques is essential to overcome these limitations and improve clinical outcomes. This manuscript presents an extensive overview of the properties and biological functions of HDACs in cancer, provides an overview of the current state of development and limitations of clinical HDAC inhibitors, and analyzes a range of innovative medicinal chemistry techniques that are applied. These techniques include selective inhibitors, dual-target inhibitors, proteolysis targeting chimeras, and protein-protein interaction inhibitors.
    Keywords:  Cancer; Drug discovery; Histone deacetylases; Novel technologies
    DOI:  https://doi.org/10.1016/j.ejmech.2023.115800
  8. Explor Target Antitumor Ther. 2023 ;4(4): 757-779
      Histone deacetylases (HDACs) are a class of zinc (Zn)-dependent metalloenzymes that are responsible for epigenetic modifications. HDACs are largely associated with histone proteins that regulate gene expression at the DNA level. This tight regulation is controlled by acetylation [via histone acetyl transferases (HATs)] and deacetylation (via HDACs) of histone and non-histone proteins that alter the coiling state of DNA, thus impacting gene expression as a downstream effect. For the last two decades, HDACs have been studied extensively and indicated in a range of diseases where HDAC dysregulation has been strongly correlated with disease emergence and progression-most prominently, cancer, neurodegenerative diseases, HIV, and inflammatory diseases. The involvement of HDACs as regulators in these biochemical pathways established them as an attractive therapeutic target. This review summarizes the drug development efforts exerted to create HDAC inhibitors (HDACis), specifically class I HDACs, with a focus on the medicinal chemistry, structural design, and pharmacology aspects of these inhibitors.
    Keywords:  Histone deacetylases; cap group; epigenetic regulation; medicinal chemistry; small-molecule inhibitors; zinc-binding group
    DOI:  https://doi.org/10.37349/etat.2023.00166
  9. Biochim Biophys Acta Mol Cell Biol Lipids. 2023 Sep 13. pii: S1388-1981(23)00118-X. [Epub ahead of print] 159394
      Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.
    Keywords:  Phosphatidylinositol; Phosphatidylinositol transfer proteins; Phospholipase C; RdgB
    DOI:  https://doi.org/10.1016/j.bbalip.2023.159394