bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2024‒07‒21
fifteen papers selected by
Lakesh Kumar, BITS Pilani



  1. bioRxiv. 2024 Jul 09. pii: 2024.07.08.602581. [Epub ahead of print]
      Toxoplasma gondii is a ubiquitous protozoan parasite that can reside long-term within hosts as intracellular tissue cysts comprised of chronic stage bradyzoites. To perturb chronic infection requires a better understanding of the cellular processes that mediate parasite persistence. Macroautophagy/autophagy is a catabolic and homeostatic pathway that is required for T. gondii chronic infection, although the molecular details of this process remain poorly understood. A key step in autophagy is the initial formation of the phagophore that sequesters cytoplasmic components and matures into a double-membraned autophagosome for delivery of the cargo to a cell's digestive organelle for degradative recycling. While T. gondii appears to have a reduced repertoire of autophagy proteins, it possesses a putative phospholipid scramblase, TgATG9. Through structural modeling and complementation assays, we show herein that TgATG9 can partially rescue bulk autophagy in atg9Δ yeast. We demonstrated the importance of TgATG9 for proper autophagosome dynamics at the subcellular level using three-dimensional live cell lattice light sheet microscopy. Conditional knockdown of TgATG9 in T. gondii after bradyzoite differentiation resulted in markedly reduced parasite viability. Together, our findings provide insights into the molecular dynamics of autophagosome biogenesis within an early-branching eukaryote and pinpoint the indispensable role of autophagy in maintaining T. gondii chronic infection.
    DOI:  https://doi.org/10.1101/2024.07.08.602581
  2. EMBO J. 2024 Jul 15.
      Cytokinetic abscission marks the final stage of cell division, during which the daughter cells physically separate through the generation of new barriers, such as the plasma membrane or cell wall. While the contractile ring plays a central role during cytokinesis in bacteria, fungi and animal cells, the process diverges in Apicomplexa. In Toxoplasma gondii, two daughter cells are formed within the mother cell by endodyogeny. The mechanism by which the progeny cells acquire their plasma membrane during the disassembly of the mother cell, allowing daughter cells to emerge, remains unknown. Here we identify and characterize five T. gondii proteins, including three protein phosphatase 2A subunits, which exhibit a distinct and dynamic localization pattern during parasite division. Individual downregulation of these proteins prevents the accumulation of plasma membrane at the division plane, preventing the completion of cellular abscission. Remarkably, the absence of cytokinetic abscission does not hinder the completion of subsequent division cycles. The resulting progeny are able to egress from the infected cells but fail to glide and invade, except in cases of conjoined twin parasites.
    Keywords:  Abscission; Cytokinesis; Cytoskeleton; Division Plane; Phosphatase
    DOI:  https://doi.org/10.1038/s44318-024-00171-9
  3. bioRxiv. 2024 Jul 01. pii: 2024.07.01.601569. [Epub ahead of print]
      Toxoplasma gondii causes widespread chronic infections that are not cured by current treatments due to inability to affect semi-dormant bradyzoite stages within tissue cysts. To identify compounds to eliminate chronic infection, we developed a HTS using a recently characterized strain of T. gondii that undergoes efficient conversion to bradyzoites in intro. Stage-specific expression of luciferase was used to selectively monitor growth inhibition of bradyzoites by the Library of Pharmacological Active Compounds, consisting of 1280 drug-like compounds. We identified 44 compounds with >50% inhibitory effects against bradyzoites, including several new highly potent compounds several of which have precedent for antimicrobial activity. Subsequent characterization of the compound Sanguinarine sulfate revealed potent and rapid killing against in vitro produced bradyzoites and bradyzoites harvested from chronically infected mice. These findings provide a platform for expanded screening and identify promising compounds for further preclinical development against T. gondii bradyzoites responsible for chronic infection.
    DOI:  https://doi.org/10.1101/2024.07.01.601569
  4. J Biol Chem. 2024 Jul 11. pii: S0021-9258(24)02066-0. [Epub ahead of print] 107565
      Citrate synthase catalyzes the first and the rate-limiting reaction of the tricarboxylic acid (TCA) cycle, producing citrate from the condensation of oxaloacetate and acetyl-coenzyme A. The parasitic protozoan Toxoplasma gondii has full TCA cycle activity but its physiological roles remain poorly understood. In this study, we identified three proteins with predicted citrate synthase (CS) activities and two of which were localized in the mitochondrion, including the 2-methylcitrate synthase (PrpC) that was thought to be involved in the 2-methylcitrate cycle, an alternative pathway for propionyl-CoA detoxification. Further analyses on the two mitochondrial enzymes showed that both had citrate synthase activity, but the catalytic efficiency of CS1 was much higher than that of PrpC. Consistently, deletion of CS1 resulted in significantly reduced flux of glucose derived carbons into TCA cycle intermediates, leading to decreased parasite growth. In contrast, disruption of PrpC had little effect. On the other hand, simultaneous disruption of both CS1 and PrpC resulted in more severe metabolic changes and growth defects than single deletion of either gene, suggesting that PrpC does contribute to citrate production under physiological conditions. Interestingly, deleting Δcs1 and Δprpc individually or in combination only mildly or negligibly affected the virulence of parasites in mice, suggesting that both enzymes are dispensable in vivo. The dispensability of CS1 and PrpC suggests that either the TCA cycle is not essential for the asexual reproduction of tachyzoites, or there are other routes of citrate supply in the parasite mitochondrion.
    Keywords:  Asexual Reproduction; Citrate Synthase; Metabolic Flexibility; Methylcitrate Synthase; TCA cycle
    DOI:  https://doi.org/10.1016/j.jbc.2024.107565
  5. PLoS Pathog. 2024 Jul 18. 20(7): e1012411
      Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0's localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0's essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.
    DOI:  https://doi.org/10.1371/journal.ppat.1012411
  6. J Transl Med. 2024 Jul 14. 22(1): 656
      NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2.
    Keywords:  Acetylation; Glycolysis; Metabolism; PKM2; Sirt2
    DOI:  https://doi.org/10.1186/s12967-024-05435-w
  7. bioRxiv. 2024 Jul 11. pii: 2024.07.10.602966. [Epub ahead of print]
      Plasmodium falciparum acetyl-CoA synthetase (PfACAS) protein is an important source of acetyl-CoA. We detected the mutations S868G and V949I in PfACAS by whole-genome sequencing analysis in some recrudescent parasites after antimalarial treatment with artesunate and dihydroartemisinin-piperaquine, suggesting that they may confer drug resistance. Using CRISPR/Cas9 technology, we engineered parasite lines carrying the PfACAS S868G and V949I mutations in two genetic backgrounds and evaluated their susceptibility to antimalarial drugs in vitro. The results demonstrated that PfACAS S868G and V949I mutations alone or in combination were not enough to provide resistance to antimalarial drugs.
    DOI:  https://doi.org/10.1101/2024.07.10.602966
  8. Nat Commun. 2024 Jul 17. 15(1): 6002
      The AMP-forming acetyl-CoA synthetase is regulated by lysine acetylation both in bacteria and eukaryotes. However, the underlying mechanism is poorly understood. The Bacillus subtilis acetyltransferase AcuA and the AMP-forming acetyl-CoA synthetase AcsA form an AcuA•AcsA complex, dissociating upon lysine acetylation of AcsA by AcuA. Crystal structures of AcsA from Chloroflexota bacterium in the apo form and in complex with acetyl-adenosine-5'-monophosphate (acetyl-AMP) support the flexible C-terminal domain adopting different conformations. AlphaFold2 predictions suggest binding of AcuA stabilizes AcsA in an undescribed conformation. We show the AcuA•AcsA complex dissociates upon acetyl-coenzyme A (acetyl-CoA) dependent acetylation of AcsA by AcuA. We discover an intrinsic phosphotransacetylase activity enabling AcuA•AcsA generating acetyl-CoA from acetyl-phosphate (AcP) and coenzyme A (CoA) used by AcuA to acetylate and inactivate AcsA. Here, we provide mechanistic insights into the regulation of AMP-forming acetyl-CoA synthetases by lysine acetylation and discover an intrinsic phosphotransacetylase allowing modulation of its activity based on AcP and CoA levels.
    DOI:  https://doi.org/10.1038/s41467-024-49952-0
  9. Front Cell Infect Microbiol. 2024 ;14 1408947
      Lysine acetylation is an evolutionarily conserved protein modification that changes protein functions and plays an essential role in many cellular processes, such as central metabolism, transcriptional regulation, chemotaxis, and pathogen virulence. It can alter DNA binding, enzymatic activity, protein-protein interactions, protein stability, or protein localization. In prokaryotes, lysine acetylation occurs non-enzymatically and by the action of lysine acetyltransferases (KAT). In enzymatic acetylation, KAT transfers the acetyl group from acetyl-CoA (AcCoA) to the lysine side chain. In contrast, acetyl phosphate (AcP) is the acetyl donor of chemical acetylation. Regardless of the acetylation type, the removal of acetyl groups from acetyl lysines occurs only enzymatically by lysine deacetylases (KDAC). KATs are grouped into three main superfamilies based on their catalytic domain sequences and biochemical characteristics of catalysis. Specifically, members of the GNAT are found in eukaryotes and prokaryotes and have a core structural domain architecture. These enzymes can acetylate small molecules, metabolites, peptides, and proteins. This review presents current knowledge of acetylation mechanisms and functional implications in bacterial metabolism, pathogenicity, stress response, translation, and the emerging topic of protein acetylation in the gut microbiome. Additionally, the methods used to elucidate the biological significance of acetylation in bacteria, such as relative quantification and stoichiometry quantification, and the genetic code expansion tool (CGE), are reviewed.
    Keywords:  acetylation; bacteria; functions; methods; protein
    DOI:  https://doi.org/10.3389/fcimb.2024.1408947
  10. Arch Pharm (Weinheim). 2024 Jul 14. e2400450
      Epigenetic modulators such as lysine-specific demethylase 1 (LSD1) and histone deacetylases (HDACs) are drug targets for cancer, neuropsychiatric disease, or inflammation, but inhibitors of these enzymes exhibit considerable side effects. For a potential local treatment with reduced systemic toxicity, we present here soft drug candidates as new LSD1 and HDAC inhibitors. A soft drug is a compound that is degraded in vivo to less active metabolites after having achieved its therapeutic function. This has been successfully applied for corticosteroids in the clinic, but soft drugs targeting epigenetic enzymes are scarce, with the HDAC inhibitor remetinostat being the only example. We have developed new methyl ester-containing inhibitors targeting LSD1 or HDACs and compared the biological activities of these to their respective carboxylic acid cleavage products. In vitro activity assays, cellular experiments, and a stability assay identified potent HDAC and LSD1 soft drug candidates that are superior to their corresponding carboxylic acids in cellular models.
    Keywords:  HDAC; LSD1; histone deacetylase; histone demethylase; soft drug
    DOI:  https://doi.org/10.1002/ardp.202400450
  11. Sci Rep. 2024 Jul 15. 14(1): 16278
      This study explores the role of SIRT2 in regulating autophagy and its interaction with AMPK in the context of acute liver failure (ALF). This study investigated the effects of SIRT2 and AMPK on autophagy in ALF mice and TAA-induced AML12 cells. The results revealed that the liver tissue in ALF model group had a lot of inflammatory cell infiltration and hepatocytes necrosis, which were reduced by SIRT2 inhibitor AGK2. In comparison to normal group, the level of SIRT2, P62, MDA, TOS in TAA group were significantly increased, which were decreased in AGK2 treatment. Compared with normal group, the expression of P-PRKAA1, Becilin1 and LC3B-II was decreased in TAA group. However, AGK2 enhanced the expression of P-PRKAA1, Becilin1 and LC3B-II in model group. Overexpression of SIRT2 in AML12 cell resulted in decreased P-PRKAA1, Becilin1 and LC3B-II level, enhanced the level of SIRT2, P62, MDA, TOS. Overexpression of PRKAA1 in AML12 cell resulted in decreased SIRT2, TOS and MDA level and triggered more autophagy. In conclusion, the data suggested the link between AMPK and SIRT2, and reveals the important role of AMPK and SIRT2 in autophagy on acute liver failure.
    DOI:  https://doi.org/10.1038/s41598-024-67102-w
  12. bioRxiv. 2024 Jul 14. pii: 2024.07.12.603313. [Epub ahead of print]
      Quiescence is a reversible cell cycle exit traditionally thought to be associated with a metabolically inactive state. Recent work in muscle cells indicates that metabolic reprogramming is associated with quiescence. Whether metabolic changes occur in cancer to drive quiescence is unclear. Using a multi-omics approach, we found that the metabolic enzyme ACSS2, which converts acetate into acetyl-CoA, is both highly upregulated in quiescent ovarian cancer cells and required for their survival. Indeed, quiescent ovarian cancer cells have increased levels of acetate-derived acetyl-CoA, confirming increased ACSS2 activity in these cells. Furthermore, either inducing ACSS2 expression or supplementing cells with acetate was sufficient to induce a reversible quiescent cell cycle exit. RNA-Seq of acetate treated cells confirmed negative enrichment in multiple cell cycle pathways as well as enrichment of genes in a published G0 gene signature. Finally, analysis of patient data showed that ACSS2 expression is upregulated in tumor cells from ascites, which are thought to be more quiescent, compared to matched primary tumors. Additionally, high ACSS2 expression is associated with platinum resistance and worse outcomes. Together, this study points to a previously unrecognized ACSS2-mediated metabolic reprogramming that drives quiescence in ovarian cancer. As chemotherapies to treat ovarian cancer, such as platinum, have increased efficacy in highly proliferative cells, our data give rise to the intriguing question that metabolically-driven quiescence may affect therapeutic response.
    DOI:  https://doi.org/10.1101/2024.07.12.603313
  13. Zhongguo Fei Ai Za Zhi. 2024 Jun 20. 27(6): 471-479
      Post translational modifications (PTMs) can change the properties of a protein by covalent addition of functional groups to one or more amino acids, and influence almost all aspects of normal cell biology and pathogenesis. Lactylation is a novel identified PTM, and has been found in both histone and non-histone proteins. Since associated with the end product of glycolysis-- lactate, lactylation modification could provide a new perspective for understanding the relationship between metabolic reprogramming and epigenetic modifications. Accumulated evidences suggest that lactylation play important roles in tumor progression and links to poor prognosis in clinical studies. Histone lactylation can affect gene expression in tumor cells and immunological cells, further promoting tumor progression and immune suppression. Lactylation on non-histone proteins can also regulate tumor progression and drug resistance. In this review, we aimed to summarize the roles of lactylation in cancer progression, microenvironment interactions and immune suppression, try to identify new molecular targets for cancer therapy and provide a new direction for combined targeted therapy and immunotherapy.
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    Keywords:  Cancer; Lactate; Lactylation
    DOI:  https://doi.org/10.3779/j.issn.1009-3419.2024.102.20
  14. NPJ Syst Biol Appl. 2024 Jul 18. 10(1): 77
      Energy metabolism is crucial for all living cells, especially during fast growth or stress scenarios. Many cancer and activated immune cells (Warburg effect) or yeasts (Crabtree effect) mostly rely on aerobic glucose fermentation leading to lactate or ethanol, respectively, to generate ATP. In recent years, several mathematical models have been proposed to explain the Warburg effect on theoretical grounds. Besides glucose, glutamine is a very important substrate for eukaryotic cells-not only for biosynthesis, but also for energy metabolism. Here, we present a minimal constraint-based stoichiometric model for explaining both the classical Warburg effect and the experimentally observed respirofermentation of glutamine (WarburQ effect). We consider glucose and glutamine respiration as well as the respective fermentation pathways. Our resource allocation model calculates the ATP production rate, taking into account enzyme masses and, therefore, pathway costs. While our calculation predicts glucose fermentation to be a superior energy-generating pathway in human cells, different enzyme characteristics in yeasts reduce this advantage, in some cases to such an extent that glucose respiration is preferred. The latter is observed for the fungal pathogen Candida albicans, which is a known Crabtree-negative yeast. Further, optimization results show that glutamine is a valuable energy source and important substrate under glucose limitation, in addition to its role as a carbon and nitrogen source of biomass in eukaryotic cells. In conclusion, our model provides insights that glutamine is an underestimated fuel for eukaryotic cells during fast growth and infection scenarios and explains well the observed parallel respirofermentation of glucose and glutamine in several cell types.
    DOI:  https://doi.org/10.1038/s41540-024-00393-x
  15. Commun Biol. 2024 Jul 19. 7(1): 884
      The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.
    DOI:  https://doi.org/10.1038/s42003-024-06579-7