bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2023‒03‒12
twelve papers selected by
Lakesh Kumar
BITS Pilani

  1. Nat Commun. 2023 Mar 10. 14(1): 1312
      Malaria-causing parasites of the Plasmodium genus undergo multiple developmental phases in the human and the mosquito hosts, regulated by various post-translational modifications. While ubiquitination by multi-component E3 ligases is key to regulate a wide range of cellular processes in eukaryotes, little is known about its role in Plasmodium. Here we show that Plasmodium berghei expresses a conserved SKP1/Cullin1/FBXO1 (SCFFBXO1) complex showing tightly regulated expression and localisation across multiple developmental stages. It is key to cell division for nuclear segregation during schizogony and centrosome partitioning during microgametogenesis. It is additionally required for parasite-specific processes including gamete egress from the host erythrocyte, as well as integrity of the apical and the inner membrane complexes (IMC) in merozoite and ookinete, two structures essential for the dissemination of these motile stages. Ubiquitinomic surveys reveal a large set of proteins ubiquitinated in a FBXO1-dependent manner including proteins important for egress and IMC organisation. We additionally demonstrate an interplay between FBXO1-dependent ubiquitination and phosphorylation via calcium-dependent protein kinase 1. Altogether we show that Plasmodium SCFFBXO1 plays conserved roles in cell division and is also important for parasite-specific processes in the mammalian and mosquito hosts.
  2. Int J Mol Sci. 2023 Mar 03. pii: 4902. [Epub ahead of print]24(5):
      Toxoplasma gondii is an obligate protozoon that can infect all warm-blooded animals including humans. T. gondii afflicts one-third of the human population and is a detriment to the health of livestock and wildlife. Thus far, traditional drugs such as pyrimethamine and sulfadiazine used to treat T. gondii infection are inadequate as therapeutics due to relapse, long treatment period, and low efficacy in parasite clearance. Novel, efficacious drugs have not been available. Lumefantrine, as an antimalarial, is effective in killing T. gondii but has no known mechanism of action. We combined metabolomics with transcriptomics to investigate how lumefantrine inhibits T. gondii growth. We identified significant alternations in transcripts and metabolites and their associated functional pathways that are attributed to lumefantrine treatment. RH tachyzoites were used to infect Vero cells for three hours and subsequently treated with 900 ng/mL lumefantrine. Twenty-four hours post-drug treatment, we observed significant changes in transcripts associated with five DNA replication and repair pathways. Metabolomic data acquired through liquid chromatography-tandem mass spectrometry (LC-MS) showed that lumefantrine mainly affected sugar and amino acid metabolism, especially galactose and arginine. To investigate whether lumefantrine damages T. gondii DNA, we conducted a terminal transferase assay (TUNEL). TUNEL results showed that lumefantrine significantly induced apoptosis in a dose-dependent manner. Taken together, lumefantrine effectively inhibited T. gondii growth by damaging DNA, interfering with DNA replication and repair, and altering energy and amino acid metabolisms.
    Keywords:  Toxoplasma gondii; lumefantrine; metabolomics; transcriptomics
  3. Diagn Microbiol Infect Dis. 2023 Jan 18. pii: S0732-8893(23)00011-1. [Epub ahead of print]105(4): 115901
      Primary Toxoplasma gondii (T. gondii) infection during pregnancy could result in congenital disease with severe clinical complications. IgM antibodies are one of the indices of primary infection. The IgG avidity index (AI) is also known to remain low for at least 3 months after primary infection. Here, we evaluated and compared the performance of T. gondii IgG avidity assays as confirmed by T. gondii IgM serostatus and number of days post-exposure. Four assays preferentially used in Japan were employed to measure the T. gondii IgG AI. Results for the T. gondii IgG AI showed good concordance, particularly in cases with a low IgG AI. This study confirms that the combination of T. gondii IgM and IgG AI tests is a reliable and suitable method for identifying T. gondii primary infections. Our study proposes the necessity of measuring the T. gondii IgG AI as an additional indicator of T. gondii primary infection.
    Keywords:  IgG avidity; IgM; Primary infection; Toxoplasma gondii
  4. Protein Sci. 2023 Mar 08. e4619
      Cystathionine γ-lyase (CGL) is a PLP-dependent enzyme that catalyzes the last step of the reverse transsulfuration route for endogenous cysteine biosynthesis. The canonical CGL-catalyzed process consists of an α,γ-elimination reaction that breaks down cystathionine into cysteine, α-ketobutyrate, and ammonia. In some species, the enzyme can alternatively use cysteine as a substrate, resulting in the production of hydrogen sulfide (H2 S). Importantly, inhibition of the enzyme and consequently of its H2 S production activity, makes multiresistant bacteria considerably more susceptible to antibiotics. Other organisms, such as Toxoplasma gondii, the causative agent of toxoplasmosis, encode a CGL enzyme (TgCGL) that almost exclusively catalyzes the canonical process, with only minor reactivity to cysteine. Interestingly, the substitution of N360 by a serine (the equivalent amino acid residue in the human enzyme) at the active site changes the specificity of TgCGL for the catalysis of cystathionine, resulting in an enzyme that can cleave both the CγS and the CβS bond of cystathionine. Based on these findings and to deepen the molecular basis underlying the enzyme-substrate specificity, we have elucidated the crystal structures of native TgCGL and the variant TgCGL-N360S from crystals grown in the presence of cystathionine, cysteine, and the inhibitor D,L-propargylglycine (PPG). Our structures reveal the binding mode of each molecule within the catalytic cavity and help explain the inhibitory behavior of cysteine and PPG. A specific inhibitory mechanism of TgCGL by PPG is proposed. This article is protected by copyright. All rights reserved.
    Keywords:  Reverse transsulfuration; Toxoplasma gondii; crystal structure; cystathionine γ-lyase; cysteine; inhibitor; propargylglycine; pyridoxal-5'-phosphate
  5. Vet Parasitol. 2023 Mar 02. pii: S0304-4017(23)00034-1. [Epub ahead of print]316 109903
      Coccidiosis, caused by Eimeria species, results in huge economic losses to the animal industry. Dinitolmide, a veterinary-approved coccidiostat, has a wide anticoccidial spectrum with no effect on host immunity. However, the mechanism of its anticoccidial effects remains unclear. Here, we used an in vitro culture system of T. gondii to explore the anti-Toxoplasma effect of dinitolmide and its underlying mechanism against coccidia. We show that dinitolmide has potent in vitro anti-Toxoplasma activity with the half-maximal effective concentration (EC50) of 3.625 µg/ml. Dinitolmide treatment significantly inhibited the viability, invasion and proliferation of T. gondii tachyzoites. The recovery experiment showed that dinitolmide can completely kill T. gondii tachyzoites after 24 h of treatment. Morphologically abnormal parasites were observed after dinitolmide exposure, including asynchronous development of daughter cells and deficiency of parasite inner and outer membrane. Further electron microscopy results showed that the drug could damage the membrane structure of T. gondii. By comparative transcriptomic analysis, we found that genes related to cell apoptosis and nitric-oxide synthase were up-regulated after dinitolmide treatment, which might be responsible for parasite cell death. Meanwhile, many Sag-related sequence (srs) genes were down-regulated after treatment, which could be closely associated with the reduction of parasite invasion and proliferation capacity. Our study indicates that the coccidiostat dinitolmide has a potent inhibitory effect on T. gondii in vitro and provides insight into the mode of action of the drug.
    Keywords:  Antiparasitic; Dinitolmide; In vitro; Membrane structure; Toxoplasma gondii
  6. Front Immunol. 2023 ;14 1125667
      Neutrophil extracellular traps (NET) formation is one important host innate defense mechanism elicited by polymorphonuclear neutrophils (PMN). NETs are composed by chromatin and proteins with microbicidal and signaling activity. So far, there is one report on Toxoplasma gondii-triggered NETs in cattle, however, exact mechanisms, including signalling pathways and dynamics governing this reaction remain largely unknown. Recently, involvement of cell cycle proteins was demonstrated for phorbol myristate acetate (PMA)-triggered human PMN-derived NETs. Here, we studied the involvement of cell cycle proteins in T. gondii-induced NETs in exposed bovine PMN. Through confocal and transmission electron microscopy we discovered that Ki-67 and lamin B1 signals are upregulated and relocated during T. gondii-induced NETosis. Nuclear membrane disruption was also observed as a hallmark of NET formation in bovine PMN confronted with viable T. gondii tachyzoites, mimicking some steps of mitosis. However, we did not observe centrosome duplication as previously described for human PMN-derived NET formation stimulated with PMA.
    Keywords:  Ki-67; NETs; PMN; bovine; cattle; cell cycle; centrosome; lamin B1
  7. ACS Chem Biol. 2023 Mar 09.
      Natural products play critical roles as antibiotics, anticancer therapeutics, and biofuels. Polyketides are a distinct natural product class of structurally diverse secondary metabolites that are synthesized by polyketide synthases (PKSs). The biosynthetic gene clusters that encode PKSs have been found across nearly all realms of life, but those from eukaryotic organisms are relatively understudied. A type I PKS from the eukaryotic apicomplexan parasite Toxoplasma gondii,TgPKS2, was recently discovered through genome mining, and the functional acyltransferase (AT) domains were found to be selective for malonyl-CoA substrates. To further characterize TgPKS2, we resolved assembly gaps within the gene cluster, which confirmed that the encoded protein consists of three distinct modules. We subsequently isolated and biochemically characterized the four acyl carrier protein (ACP) domains within this megaenzyme. We observed self-acylation─or substrate acylation without an AT domain─for three of the four TgPKS2 ACP domains with CoA substrates. Furthermore, CoA substrate specificity and kinetic parameters were determined for all four unique ACPs. TgACP2-4 were active with a wide scope of CoA substrates, while TgACP1 from the loading module was found to be inactive for self-acylation. Previously, self-acylation has only been observed in type II systems, which are enzymes that act in-trans with one another, and this represents the first report of this activity in a modular type I PKS whose domains function in-cis. Site-directed mutagenesis of specific TgPKS2 ACP3 acidic residues near the phosphopantetheinyl arm demonstrated that they influence self-acylation activity and substrate specificity, possibly by influencing substrate coordination or phosphopantetheinyl arm activation. Further, the lack of TgPKS2 ACP self-acylation with acetoacetyl-CoA, which is utilized by previously characterized type II PKS systems, suggests that the substrate carboxyl group may be critical for TgPKS2 ACP self-acylation. The unexpected properties observed from T. gondii PKS ACP domains highlight their distinction from well-characterized microbial and fungal systems. This work expands our understanding of ACP self-acylation beyond type II systems and helps pave the way for future studies on biosynthetic enzymes from eukaryotes.
  8. Int J Mol Sci. 2023 Mar 03. pii: 4912. [Epub ahead of print]24(5):
      The accessibility of DNA to different cellular functions requires a dynamic regulation of chromatin organization that is mediated by different epigenetic modifications, which regulate chromatin accessibility and degree of compaction. These epigenetic modifications, particularly the acetylation of histone H4 in lysine 14 (H4K16ac), determine the degree of chromatin accessibility to different nuclear functions, as well as to DNA damage drugs. H4K16ac is regulated by the balance between two alternative histone modifications, acetylation and deacetylation, which are mediated by acetylases and deacetylases. Tip60/KAT5 acetylates, and SIRT2 deacetylates histone H4K16. However, the balance between these two epigenetic enzymes is unknown. VRK1 regulates the level of H4K16 acetylation by activating Tip60. We have shown that the VRK1 and SIRT2 are able to form a stable protein complex. For this work, we used in vitro interaction, pull-down and in vitro kinase assays. In cells, their interaction and colocalization were detected by immunoprecipitation and immunofluorescence. The kinase activity of VRK1 is inhibited by a direct interaction of its N-terminal kinase domain with SIRT2 in vitro. This interaction causes a loss of H4K16ac similarly to the effect of a novel VRK1 inhibitor (VRK-IN-1) or VRK1 depletion. The use of specific SIRT2 inhibitors in lung adenocarcinoma cells induces H4K16ac, contrary to the novel VRK-IN-1 inhibitor, which prevents H4K16ac and a correct DNA damage response. Therefore, the inhibition of SIRT2 can cooperate with VRK1 in the accessibility of drugs to chromatin in response to DNA damage caused by doxorubicin.
    Keywords:  DNA damage response; KAT5; SIRT2; Tip60; VRK-IN-1; VRK1; acetylation; histone H4
  9. PLoS Negl Trop Dis. 2023 Mar 10. 17(3): e0011191
      Trypanosoma cruzi cAMP-mediated invasion has long been described, however, the detailed mechanism of action of the pathway activated by this cyclic nucleotide still remains unknown. We have recently demonstrated a crucial role for Epac in the cAMP-mediated invasion of the host cell. In this work, we gathered evidence indicating that the cAMP/Epac pathway is activated in different cells lines. In accordance, data collected from pull-down experiments designed to identify only the active form of Rap1b (Rap1b-GTP), and infection assays using cells transfected with a constitutively active mutant of Rap1b (Rap1b-G12V), strongly suggest the participation of Rap1b as mediator of the pathway. In addition to the activation of this small GTPase, fluorescence microscopy allowed us to demonstrate the relocalization of Rap1b to the entry site of the parasite. Moreover, phospho-mimetic and non-phosphorylable mutants of Rap1b were used to demonstrate a PKA-dependent antagonistic effect on the pathway, by phosphorylation of Rap1b, and potentially of Epac. Finally, Western Blot analysis was used to determine the involvement of the MEK/ERK signalling downstream of cAMP/Epac/Rap1b-mediated invasion.
  10. Brief Bioinform. 2023 Mar 10. pii: bbad090. [Epub ahead of print]
      Protein phosphorylation, one of the main protein post-translational modifications, is required for regulating various life activities. Kinases and phosphatases that regulate protein phosphorylation in humans have been targeted to treat various diseases, particularly cancer. High-throughput experimental methods to discover protein phosphosites are laborious and time-consuming. The burgeoning databases and predictors provide essential infrastructure to the research community. To date, >60 publicly available phosphorylation databases and predictors each have been developed. In this review, we have comprehensively summarized the status and applicability of major online phosphorylation databases and predictors, thereby helping researchers rapidly select tools that are most suitable for their projects. Moreover, the organizational strategies and limitations of these databases and predictors have been highlighted, which may facilitate the development of better protein phosphorylation predictors in silico.
    Keywords:  database; post-translational modification; predictor; protein phosphorylation
  11. J Cell Biol. 2023 May 01. pii: e202109162. [Epub ahead of print]222(5):
      Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.
  12. Future Med Chem. 2023 Mar 09.
      The silent information regulator (sirtuin) is a family of enzymes involved in epigenetic processes with lysine deacetylase activity, having as substrates histones and other proteins. They participate in a wide range of cellular and pathologic processes, such as gene expression, cell division and motility, oxidative-induced stress management, metabolic control and carcinogenesis, among others, thus presenting as interesting therapeutic targets. In this article, the authors describe the inhibitory mechanisms and binding modes of the human sirtuin 2 (hSIRT2) inhibitors, which had their complexes with the enzyme structurally characterized. The results help pave the way for the rational designing of new hSIRT2 inhibitors and the development of novel therapeutic agents targeting this epigenetic enzyme.
    Keywords:  HDAC; drug design; enzyme inhibition; enzyme inhibitory mechanisms; epigenetics; hSIRT2; histone deacetylases; ligand binding modes; lysine deacetylases; sirtuin 2