bims-toxgon 2023-04-09 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–04–09
eleven papers selected by
Lakesh Kumar, BITS Pilani

Pantothenate biosynthesis in Toxoplasma gondii tachyzoites is not a drug target.
Computational screening of FDA-approved drugs to identify potential TgDHFR, TgPRS, and TgCDPK1 proteins inhibitors against Toxoplasma gondii.
Toxoplasma ERK7 protects the apical complex from premature degradation.
Proximity-Dependent Biotinylation and Identification of Flagellar Proteins in Trypanosoma cruzi.
Serous macular detachment in ocular toxoplasmosis: A review.
Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii.
A novel Toxoplasma gondii TGGT1_316290 mRNA-LNP vaccine elicits protective immune response against toxoplasmosis in mice.
Modulation of plant acetyl-CoA synthetase activity by post-translational lysine acetylation.
Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation.
State-of-the-art techniques to study epigenetics.
Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

Int J Parasitol Drugs Drug Resist. 2023 Mar 15. pii: S2211-3207(23)00011-8. [Epub ahead of print]22 1-8

Pantothenate biosynthesis in Toxoplasma gondii tachyzoites is not a drug target.

Vanessa M Howieson, Joy Zeng, Joachim Kloehn, Christina Spry, Chiara Marchetti, Matteo Lunghi, Emmanuel Varesio, Andrew Soper, Anthony G Coyne, Chris Abell, Giel G van Dooren, Kevin J Saliba.

  Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.

Keywords:  PanC; Pantoate β-alanine ligase; Pantothenate; Pantothenate synthetase; SW314; T. gondii

DOI:  https://doi.org/10.1016/j.ijpddr.2023.03.003
Sci Rep. 2023 Apr 03. 13(1): 5396

Computational screening of FDA-approved drugs to identify potential TgDHFR, TgPRS, and TgCDPK1 proteins inhibitors against Toxoplasma gondii.

Zahra Gharibi, Behzad Shahbazi, Hamed Gouklani, Hoda Nassira, Zahra Rezaei, Khadijeh Ahmadi.

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world, because about a third of the world's population is seropositive for toxoplasmosis. Treatment regimens for toxoplasmosis have remained unchanged for the past 20 years, and no new drugs have been introduced to the market recently. This study, performed molecular docking to identify interactions of FDA-approved drugs with essential residues in the active site of proteins of T. gondii Dihydrofolate Reductase (TgDHFR), Prolyl-tRNA Synthetase (TgPRS), and Calcium-Dependent Protein Kinase 1 (TgCDPK1). Each protein was docked with 2100 FDA-approved drugs using AutoDock Vina. Also, the Pharmit software was used to generate pharmacophore models based on the TgDHFR complexed with TRC-2533, TgPRS in complex with halofuginone, and TgCDPK1 in complex with a bumped kinase inhibitor, RM-1-132. Molecular dynamics (MD) simulation was also performed for 100 ns to verify the stability of interaction in drug-protein complexes. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis evaluated the binding energy of selected complexes. Ezetimibe, Raloxifene, Sulfasalazine, Triamterene, and Zafirlukast drugs against the TgDHFR protein, Cromolyn, Cefexim, and Lactulose drugs against the TgPRS protein, and Pentaprazole, Betamethasone, and Bromocriptine drugs against TgCDPK1 protein showed the best results. These drugs had the lowest energy-based docking scores and also stable interactions based on MD analyses with TgDHFR, TgPRS, and TgCDPK1 drug targets that can be introduced as possible drugs for laboratory investigations to treat T. gondii parasite infection.

DOI: https://doi.org/10.1038/s41598-023-32388-9
J Cell Biol. 2023 Jun 05. pii: e202209098. [Epub ahead of print]222(6):

Toxoplasma ERK7 protects the apical complex from premature degradation.

William J O'Shaughnessy, Xiaoyu Hu, Sarah Ana Henriquez, Michael L Reese.

Accurate cellular replication balances the biogenesis and turnover of complex structures. In the apicomplexan parasite Toxoplasma gondii, daughter cells form within an intact mother cell, creating additional challenges to ensuring fidelity of division. The apical complex is critical to parasite infectivity and consists of apical secretory organelles and specialized cytoskeletal structures. We previously identified the kinase ERK7 as required for maturation of the apical complex in Toxoplasma. Here, we define the Toxoplasma ERK7 interactome, including a putative E3 ligase, CSAR1. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by mislocalization from the parasite residual body to the apical complex. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.

DOI: https://doi.org/10.1083/jcb.202209098
mSphere. 2023 Apr 05. e0008823

Proximity-Dependent Biotinylation and Identification of Flagellar Proteins in Trypanosoma cruzi.

Madalyn M Won, Aaron Baublis, Barbara A Burleigh.

  The flagellated kinetoplastid protozoan and causative agent of human Chagas disease, Trypanosoma cruzi, inhabits both invertebrate and mammalian hosts over the course of its complex life cycle. In these disparate environments, T. cruzi uses its single flagellum to propel motile life stages and, in some instances, to establish intimate contact with the host. Beyond its role in motility, the functional capabilities of the T. cruzi flagellum have not been defined. Moreover, the lack of proteomic information for this organelle, in any parasite life stage, has limited functional investigation. In this study, we employed a proximity-dependent biotinylation approach based on the differential targeting of the biotin ligase TurboID to the flagellum or cytosol in replicative stages of T. cruzi to identify proteins that are enriched in the flagellum by mass spectrometry. Proteomic analysis of the resulting biotinylated protein fractions yielded 218 candidate flagellar proteins in T. cruzi epimastigotes (insect stage) and 99 proteins in intracellular amastigotes (mammalian stage). Forty of these enriched flagellar proteins were common to both parasite life stages and included orthologs of known flagellar proteins in other trypanosomatid species, proteins specific to the T. cruzi lineage and hypothetical proteins. With the validation of flagellar localization for several of the identified candidates, our results demonstrate that TurboID-based proximity proteomics is an effective tool for probing subcellular compartments in T. cruzi. The proteomic data sets generated in this work offer a valuable resource to facilitate functional investigation of the understudied T. cruzi flagellum. IMPORTANCE Trypanosoma cruzi is a protozoan parasite that causes Chagas disease, which causes substantial morbidity and mortality in South and Central America. Throughout its life cycle, T. cruzi interacts with insect and mammalian hosts via its single flagellum, establishing intimate contact with host membranes. Currently, few flagellar proteins have been identified in T. cruzi that could provide insight into the mechanisms involved in mediating physical and biochemical interactions with the host. Here, we set out to identify flagellar proteins in the main replicative stages of T. cruzi using a proximity-labeling approach coupled with mass spectrometry. The >200 candidate flagellar proteins identified represent the first large-scale identification of candidate flagellar proteins in T. cruzi with preliminary validation. These data offer new avenues to investigate the biology of T. cruzi-host interactions, a promising area for development of new strategies aimed at the control of this pathogen.

Keywords:  Trypanosoma cruzi; TurboID; amastigote; flagellum; proteomics; proximity labeling

DOI:  https://doi.org/10.1128/msphere.00088-23
Eur J Ophthalmol. 2023 Apr 04. 11206721231168148

Serous macular detachment in ocular toxoplasmosis: A review.

Daniele Cirone, Francesco Pellegrini, Alessandra Cuna, Ettore Caruso, Luca Cimino, Francesca Leonardi.

  Ocular toxoplasmosis, a disease of the eye caused by the protozoan parasite Toxoplasma gondii, represents a common cause of posterior uveitis. The Authors review the current Literature regarding the uncommon presentation of ocular toxoplasmosis as macular serous retinal detachment (SRD). It is imperative to keep in mind that inflammatory SRD is a possible presentation of toxoplasmic retinochoroiditis. Underestimation of this clinical scenario and treatment with steroids alone without appropriate antiparasitic drugs, could lead to devastating consequences.

Keywords:  Toxoplasma gondii; ocular toxoplasmosis; serous retinal detachment; uveitis

DOI:  https://doi.org/10.1177/11206721231168148
Elife. 2023 Apr 04. pii: e86049. [Epub ahead of print]12

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii.

Amit Kumar, Oscar Vadas, Nicolas Dos Santos Pacheco, Xu Zhang, Kin Chao, Nicolas Darvill, Helena Ø Rasmussen, Yingqi Xu, Gloria Meng-Hsuan Lin, Fisentzos A Stylianou, Jan Skov Pedersen, Sarah L Rouse, Marc L Morgan, Dominique Soldati-Favre, Stephen Matthews.

  The phylum of Apicomplexa groups intracellular parasites that employ substrate-dependent gliding motility to invade host cells, egress from the infected cells and cross biological barriers. The glideosome associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the solution properties together with membrane and F-actin binding interfaces suggest that GAC adopts several conformations from closed to open and extended. A multi-conformational model for assembly and regulation of GAC within the glideosome is proposed.

Keywords:  P. falciparum; human; infectious disease; microbiology; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.86049
Front Microbiol. 2023 ;14 1145114

A novel Toxoplasma gondii TGGT1_316290 mRNA-LNP vaccine elicits protective immune response against toxoplasmosis in mice.

Dan Li, Yizhuo Zhang, Shiyu Li, Bin Zheng.

  Toxoplasma gondii (T. gondii) can infect almost all warm-blooded animals and is a major threat to global public health. Currently, there is no effective drug or vaccine for T. gondii. In this study, bioinformatics analysis on B and T cell epitopes revealed that TGGT1_316290 (TG290) had superior effects compared with the surface antigen 1 (SAG1). TG290 mRNA-LNP was constructed through the Lipid Nanoparticle (LNP) technology and intramuscularly injected into the BALB/c mice, and its immunogenicity and efficacy were explored. Analysis of antibodies, cytokines (IFN-γ, IL-12, IL-4, and IL-10), lymphocytes proliferation, cytotoxic T lymphocyte activity, dendritic cell (DC) maturation, as well as CD4+ and CD8+ T lymphocytes revealed that TG290 mRNA-LNP induced humoral and cellular immune responses in vaccinated mice. Furthermore, T-Box 21 (T-bet), nuclear factor kappa B (NF-kB) p65, and interferon regulatory factor 8 (IRF8) subunit were over-expressed in the TG290 mRNA-LNP-immunized group. The survival time of mice injected with TG290 mRNA-LNP was significantly longer (18.7 ± 3 days) compared with the survival of mice of the control groups (p < 0.0001). In addition, adoptive immunization using 300 μl serum and lymphocytes (5*107) of mice immunized with TG290 mRNA-LNP significantly prolonged the survival time of these mice. This study demonstrates that TG290 mRNA-LNP induces specific immune response against T. gondii and may be a potential toxoplasmosis vaccine candidate for this infection.

Keywords:  TGGT1_316290; Toxoplasma gondii; immune response; lipid nanoparticle; mRNA vaccine

DOI:  https://doi.org/10.3389/fmicb.2023.1145114
Front Mol Biosci. 2023 ;10 1117921

Modulation of plant acetyl-CoA synthetase activity by post-translational lysine acetylation.

Naazneen Sofeo, Dirk C Winkelman, Karina Leung, Basil J Nikolau.

  Acetyl-CoA synthetase (ACS) is one of several enzymes that generate the key metabolic intermediate, acetyl-CoA. In microbes and mammals ACS activity is regulated by the post-translational acetylation of a key lysine residue. ACS in plant cells is part of a two-enzyme system that maintains acetate homeostasis, but its post-translational regulation is unknown. This study demonstrates that the plant ACS activity can be regulated by the acetylation of a specific lysine residue that is positioned in a homologous position as the microbial and mammalian ACS sequences that regulates ACS activity, occurring in the middle of a conserved motif, near the carboxyl-end of the protein. The inhibitory effect of the acetylation of residue Lys-622 of the Arabidopsis ACS was demonstrated by site-directed mutagenesis of this residue, including its genetic substitution with the non-canonical N-ε-acetyl-lysine residue. This latter modification lowered the catalytic efficiency of the enzyme by a factor of more than 500-fold. Michaelis-Menten kinetic analysis of the mutant enzyme indicates that this acetylation affects the first half-reaction of the ACS catalyzed reaction, namely, the formation of the acetyl adenylate enzyme intermediate. The post-translational acetylation of the plant ACS could affect acetate flux in the plastids and overall acetate homeostasis.

Keywords:  acetyl-CoA synthetase; acetylation; arabidopsis; genetic code expansion; post-translation modification; regulation

DOI:  https://doi.org/10.3389/fmolb.2023.1117921
BMC Biol. 2023 Apr 03. 21(1): 65

Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation.

Myriam D Jeninga, Jingyi Tang, Shamista A Selvarajah, Alexander G Maier, Michael F Duffy, Michaela Petter.

   BACKGROUND: The protozoan malaria parasite Plasmodium falciparum has a complex life cycle during which it needs to differentiate into multiple morphologically distinct life forms. A key process for transmission of the disease is the development of male and female gametocytes in the human blood, yet the mechanisms determining sexual dimorphism in these haploid, genetically identical sexual precursor cells remain largely unknown. To understand the epigenetic program underlying the differentiation of male and female gametocytes, we separated the two sexual forms by flow cytometry and performed RNAseq as well as comprehensive ChIPseq profiling of several histone variants and modifications.
RESULTS: We show that in female gametocytes the chromatin landscape is globally remodelled with respect to genome-wide patterns and combinatorial usage of histone variants and histone modifications. We identified sex specific differences in heterochromatin distribution, implicating exported proteins and ncRNAs in sex determination. Specifically in female gametocytes, the histone variants H2A.Z/H2B.Z were highly enriched in H3K9me3-associated heterochromatin. H3K27ac occupancy correlated with stage-specific gene expression, but in contrast to asexual parasites this was unlinked to H3K4me3 co-occupancy at promoters in female gametocytes.
CONCLUSIONS: Collectively, we defined novel combinatorial chromatin states differentially organising the genome in gametocytes and asexual parasites and unravelled fundamental, sex-specific differences in the epigenetic code. Our chromatin maps represent an important resource for future understanding of the mechanisms driving sexual differentiation in P. falciparum.

Keywords:  Epigenome; Gametocytogenesis; Histone variants; Malaria; Plasmodium falciparum; Transcriptome

DOI:  https://doi.org/10.1186/s12915-023-01568-4
Prog Mol Biol Transl Sci. 2023 ;pii: S1877-1173(23)00038-8. [Epub ahead of print]197 23-50

State-of-the-art techniques to study epigenetics.

Ashok Saini, Yashaswi Rawat, Kritika Jain, Indra Mani.

  The epigenome consists of all the epigenetic alterations like DNA methylation, the histone modifications and non-coding RNAs which change the gene expression and have a role in diseases like cancer and other processes. Epigenetic modifications can control gene expression through variable gene activity at various levels which affects various cellular phenomenon such as cell differentiations, variability, morphogenesis, and the adaptability of an organism. Various factors such as food, pollutants, drugs, stress etc., impact the epigenome. Epigenetic mechanisms mainly involve various post-translational alteration of histones and DNA methylation. Numerous methods have been utilized to study these epigenetic marks. Various histone modifications and binding of histone modifier proteins can be analyzed using chromatin immunoprecipitation (ChIP) which is one of broadly utilized method. Other modified forms of the ChIP have been developed such as reverse chromatin immunoprecipitation (R-ChIP); sequential ChIP (ChIP-re-ChIP) and some high-throughput modified forms of ChIP such as ChIP-seq and ChIP-on-chip. Another epigenetic mechanism is DNA methylation, in which DNA methyltransferases (DNMTs) add a methyl group to the C-5 position of the cytosine. Bisulfite sequencing is the oldest and usually utilized method to measure the DNA methylation status. Other techniques have been established are whole genome bisulfite sequencing (WGBS), methylated DNA immune-precipitation based methods (MeDIP), methylation sensitive restriction enzyme digestion followed by sequencing (MRE-seq) and methylation BeadChip to study the methylome. This chapter briefly discusses the key principles and methods used to study epigenetics in health and disease conditions.

Keywords:  ChiP; DNA methylation; Epigenetics; Histone acetylation; Non-coding RNA

DOI:  https://doi.org/10.1016/bs.pmbts.2023.02.004
Front Endocrinol (Lausanne). 2023 ;14 1148412

Application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

Hui Meng, Manman Nan, Yizhen Li, Yi Ding, Yuhui Yin, Mingzhi Zhang.

  Colon cancer is the fourth leading cause of cancer death worldwide, and its progression is accompanied by a complex array of genetic variations. CRISPR/Cas9 can identify new drug-resistant or sensitive mutations in colon cancer, and can use gene editing technology to develop new therapeutic targets and provide personalized treatments, thereby significantly improving the treatment of colon cancer patients. CRISPR/Cas9 systems are driving advances in biotechnology. RNA-directed Cas enzymes have accelerated the pace of basic research and led to clinical breakthroughs. This article reviews the rapid development of CRISPR/Cas in colon cancer, from gene editing to transcription regulation, gene knockout, genome-wide CRISPR tools, therapeutic targets, stem cell genomics, immunotherapy, metabolism-related genes and inflammatory bowel disease. In addition, the limitations and future development of CRISPR/Cas9 in colon cancer studies are reviewed. In conclusion, this article reviews the application of CRISPR-Cas9 gene editing technology in basic research, diagnosis and treatment of colon cancer.

Keywords:  CRISPR tools; Genetic tool; basic research; colorectal carcinoma; genome editing

DOI:  https://doi.org/10.3389/fendo.2023.1148412