bims-toxgon 2023-02-26 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–02–26
twelve papers selected by
Lakesh Kumar, BITS Pilani

Converting and hoarding driven by protein phosphorylation in Toxoplasma gondii.
Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness.
Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum.
The Toxoplasma micropore mediates endocytosis for selective nutrient salvage from host cell compartments.
Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses.
Interplays of AMPK and TOR in Autophagy Regulation in Yeast.
Nano-Encapsulated Antioxidant: Retinoic Acid as a Natural Mucosal Adjuvant for Intranasal Immunization against Chronic Experimental Toxoplasmosis.
Proximity-dependent biotinylation and identification of flagellar proteins in Trypanosoma cruzi.
IL-18BP mediates the balance between protective and pathological immune responses to Toxoplasma gondii.
A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency.
Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.
Implications of altered sirtuins in metabolic regulation and oral cancer.

Trends Parasitol. 2023 Feb 17. pii: S1471-4922(23)00031-4. [Epub ahead of print]

Converting and hoarding driven by protein phosphorylation in Toxoplasma gondii.

Martin Blume, Deborah Maus.

  Successful parasitism relies on the evasion of adversarial host responses. Wang et al. have recently shown that Toxoplasma gondii relies on the protein phosphatase 2A (PP2A) to cause persisting infections. The phosphatase controls the development of dormant parasite stages and the accumulation of sugar supplies.

Keywords:  Toxoplasma gondii; bradyzoites; metabolism; protein phosphatase

DOI:  https://doi.org/10.1016/j.pt.2023.02.002
bioRxiv. 2023 Feb 14. pii: 2023.02.14.528480. [Epub ahead of print]

Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness.

Laura Vanagas, Daniela Muñoz, Constanza Cristaldi, Agustina Ganuza, Rosario Nájera, Mabel C Bonardi, Valeria R Turowski, Fanny Guzman, Bin Deng, Kami Kim, William J Sullivan, Sergio O Angel.

Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by "reader" proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c- Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc- R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N- terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.

DOI: https://doi.org/10.1101/2023.02.14.528480
Parasitol Res. 2023 Feb 18.

Analytical sensitivity of a multiplex quantitative PCR for Toxoplasma gondii and Neospora caninum.

Marcus Truong, Jan Šlapeta.

  Cyst-forming coccidia, Toxoplasma gondii and Neospora caninum, are recognised as important causes of animal disease. Molecular diagnostics based on the presence of DNA in animal tissue are required to specifically detect T. gondii and N. caninum while achieving high levels of analytical sensitivity. We optimised available single-plex probe base qPCR assays into a multiplexed qPCR panel to detect cyst-forming coccidia, i.e. T. gondii and N. caninum. The T. gondii assay is based on a 529-bp repetitive (REP) element and the N. caninum assay on the NC5 repetitive region. Using target sequence synthetic DNA, the limit of detection (LOD) was determined to be 100 copies, that is less than a single tachyzoite of either T. gondii or N. caninum. The T. gondii and N. caninum multiplexed qPCR assay optimised in this study can be used to effectively detect parasite DNA for diagnostic purposes in animal tissue.

Keywords:  Cyst-forming coccidia; DNA; Diagnostics; Hammondia; Neosporosis; Real-time PCR; Toxoplasmosis

DOI:  https://doi.org/10.1007/s00436-023-07796-5
Nat Commun. 2023 Feb 22. 14(1): 977

The Toxoplasma micropore mediates endocytosis for selective nutrient salvage from host cell compartments.

Wenyan Wan, Hui Dong, De-Hua Lai, Jiong Yang, Kai He, Xiaoyan Tang, Qun Liu, Geoff Hide, Xing-Quan Zhu, L David Sibley, Zhao-Rong Lun, Shaojun Long.

Apicomplexan parasite growth and replication relies on nutrient acquisition from host cells, in which intracellular multiplication occurs, yet the mechanisms that underlie the nutrient salvage remain elusive. Numerous ultrastructural studies have documented a plasma membrane invagination with a dense neck, termed the micropore, on the surface of intracellular parasites. However, the function of this structure remains unknown. Here we validate the micropore as an essential organelle for endocytosis of nutrients from the host cell cytosol and Golgi in the model apicomplexan Toxoplasma gondii. Detailed analyses demonstrated that Kelch13 is localized at the dense neck of the organelle and functions as a protein hub at the micropore for endocytic uptake. Intriguingly, maximal activity of the micropore requires the ceramide de novo synthesis pathway in the parasite. Thus, this study provides insights into the machinery underlying acquisition of host cell-derived nutrients by apicomplexan parasites that are otherwise sequestered from host cell compartments.

DOI: https://doi.org/10.1038/s41467-023-36571-4
mBio. 2023 Feb 21. e0286822

Late Embryogenesis Abundant Proteins Contribute to the Resistance of Toxoplasma gondii Oocysts against Environmental Stresses.

David Arranz-Solís, David Warschkau, Benedikt T Fabian, Frank Seeber, Jeroen P J Saeij.

  Toxoplasma gondii oocysts, which are shed in large quantities in the feces from infected felines, are very stable in the environment, resistant to most inactivation procedures, and highly infectious. The oocyst wall provides an important physical barrier for sporozoites contained inside oocysts, protecting them from many chemical and physical stressors, including most inactivation procedures. Furthermore, sporozoites can withstand large temperature changes, even freeze-thawing, as well as desiccation, high salinity, and other environmental insults; however, the genetic basis for this environmental resistance is unknown. Here, we show that a cluster of four genes encoding Late Embryogenesis Abundant (LEA)-related proteins are required to provide Toxoplasma sporozoites resistance to environmental stresses. Toxoplasma LEA-like genes (TgLEAs) exhibit the characteristic features of intrinsically disordered proteins, explaining some of their properties. Our in vitro biochemical experiments using recombinant TgLEA proteins show that they have cryoprotective effects on the oocyst-resident lactate dehydrogenase enzyme and that induced expression in E. coli of two of them leads to better survival after cold stress. Oocysts from a strain in which the four LEA genes were knocked out en bloc were significantly more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts. We discuss the evolutionary acquisition of LEA-like genes in Toxoplasma and other oocyst-producing apicomplexan parasites of the Sarcocystidae family and discuss how this has likely contributed to the ability of sporozoites within oocysts to survive outside the host for extended periods. Collectively, our data provide a first molecular detailed view on a mechanism that contributes to the remarkable resilience of oocysts against environmental stresses. IMPORTANCE Toxoplasma gondii oocysts are highly infectious and may survive in the environment for years. Their resistance against disinfectants and irradiation has been attributed to the oocyst and sporocyst walls by acting as physical and permeability barriers. However, the genetic basis for their resistance against stressors like changes in temperature, salinity, or humidity, is unknown. We show that a cluster of four genes encoding Toxoplasma Late Embryogenesis Abundant (TgLEA)-related proteins are important for this resistance to environmental stresses. TgLEAs have features of intrinsically disordered proteins, explaining some of their properties. Recombinant TgLEA proteins show cryoprotective effects on the parasite's lactate dehydrogenase, an abundant enzyme in oocysts, and expression in E. coli of two TgLEAs has a beneficial effect on growth after cold stress. Moreover, oocysts from a strain lacking all four TgLEA genes were more susceptible to high salinity, freezing, and desiccation compared to wild-type oocysts, highlighting the importance of the four TgLEAs for oocyst resilience.

Keywords:  Toxoplasma gondii; environmental resistance; intrinsically disordered proteins; late embryogenesis abundant proteins; oocysts; sporozoite

DOI:  https://doi.org/10.1128/mbio.02868-22
Cells. 2023 Feb 04. pii: 519. [Epub ahead of print]12(4):

Interplays of AMPK and TOR in Autophagy Regulation in Yeast.

John-Patrick Alao, Luc Legon, Aleksandra Dabrowska, Anne-Marie Tricolici, Juhi Kumar, Charalampos Rallis.

  Cells survey their environment and need to balance growth and anabolism with stress programmes and catabolism towards maximum cellular bioenergetics economy and survival. Nutrient-responsive pathways, such as the mechanistic target of rapamycin (mTOR) interact and cross-talk, continuously, with stress-responsive hubs such as the AMP-activated protein kinase (AMPK) to regulate fundamental cellular processes such as transcription, protein translation, lipid and carbohydrate homeostasis. Especially in nutrient stresses or deprivations, cells tune their metabolism accordingly and, crucially, recycle materials through autophagy mechanisms. It has now become apparent that autophagy is pivotal in lifespan, health and cell survival as it is a gatekeeper of clearing damaged macromolecules and organelles and serving as quality assurance mechanism within cells. Autophagy is hard-wired with energy and nutrient levels as well as with damage-response, and yeasts have been instrumental in elucidating such connectivities. In this review, we briefly outline cross-talks and feedback loops that link growth and stress, mainly, in the fission yeast Schizosaccharomyces pombe, a favourite model in cell and molecular biology.

Keywords:  S. pombe; ageing; caloric restriction; fission yeast; lifespan; mTOR; rapamycin

DOI:  https://doi.org/10.3390/cells12040519
Trop Med Infect Dis. 2023 Feb 07. pii: 106. [Epub ahead of print]8(2):

Nano-Encapsulated Antioxidant: Retinoic Acid as a Natural Mucosal Adjuvant for Intranasal Immunization against Chronic Experimental Toxoplasmosis.

Doaa E Said, Eglal I Amer, Eman Sheta, Shaimaa Makled, Fadwa M Arafa, Hala E Diab.

  The tight relationship between immunity and retinoid levels provides evidence on the critical role of retinoic acid (RA) in regulating immune activity, especially the mucosal one. Mucosal immune response is the key for determination of the outcome of infection, particularly against intracellular mucosal pathogens such as Toxoplasma gondii, where it plays a crucial role as a sentinel against parasite invasion. Herein, the immunomodulatory adjuvant role of RA was evaluated for prophylactic vaccination against chronic Toxoplasma infection. A quantity of 15 µg of RA pre-encapsulated with lipid-based nanoparticles (SLNs) was intranasally used in three doses, two weeks apart, as an adjuvant to the Toxoplasma lysate antigen (TLA). Afterward, mice were infected with 20 cysts of T. gondii (ME49 strain) and were sacrificed at the 4th week post-infection. Parasitological, immunological, biochemical, and histopathological studies were applied as vaccine efficacy measures. The protective role of the tested vaccine was noted using the statistically marked reduction in brain cyst count, accompanied by remarkable levels of protective IFN-γ and antibodies, with amelioration of infection-induced oxidative stress and brain pathology. Ultimately, this experiment outlined the prospective role of a novel, natural, nano-encapsulated and mucosal vaccine adjuvant RA-SLNs as a propitious candidate against chronic toxoplasmosis.

Keywords:  IgA; Toxoplasma gondii; adjuvant; mucosal; retinoic acid; vaccine

DOI:  https://doi.org/10.3390/tropicalmed8020106
bioRxiv. 2023 Feb 22. pii: 2023.02.16.528900. [Epub ahead of print]

Proximity-dependent biotinylation and identification of flagellar proteins in Trypanosoma cruzi.

Madalyn M Won, Aaron Baublis, Barbara Anne 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 flagellar-enriched proteins 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 flagellar-enriched 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 datasets generated in this work offer a valuable resource to facilitate functional investigation of the understudied T. cruzi flagellum.

DOI: https://doi.org/10.1101/2023.02.16.528900
Cell Rep. 2023 Feb 22. pii: S2211-1247(23)00158-4. [Epub ahead of print]42(3): 112147

IL-18BP mediates the balance between protective and pathological immune responses to Toxoplasma gondii.

Joseph T Clark, Orr-El Weizman, Daniel L Aldridge, Lindsey A Shallberg, Julia Eberhard, Zachary Lanzar, Devon Wasche, John D Huck, Ting Zhou, Aaron M Ring, Christopher A Hunter.

  Interleukin-18 (IL-18) promotes natural killer (NK) and T cell production of interferon (IFN)-γ, a key factor in resistance to Toxoplasma gondii, but previous work has shown a limited role for endogenous IL-18 in control of this parasite. Although infection with T. gondii results in release of IL-18, the production of IFN-γ induces high levels of the IL-18 binding protein (IL-18BP). Antagonism of IL-18BP with a "decoy-to-the-decoy" (D2D) IL-18 construct that does not signal but rather binds IL-18BP results in enhanced innate lymphoid cell (ILC) and T cell responses and improved parasite control. In addition, the use of IL-18 resistant to IL-18BP ("decoy-resistant" IL-18 [DR-18]) is more effective than exogenous IL-18 at promoting innate resistance to infection. DR-18 enhances CD4+ T cell production of IFN-γ but results in CD4+ T cell-mediated pathology. Thus, endogenous IL-18BP restrains aberrant immune pathology, and this study highlights strategies that can be used to tune this regulatory pathway for optimal anti-pathogen responses.

Keywords:  CD4 T cells; CD8 T cell; CP: Immunology; CP: Microbiology; IFN-gamma; IL-18; IL-18 binding protein; NK cell; Toxoplasma; cytokine; infection; pathology

DOI:  https://doi.org/10.1016/j.celrep.2023.112147
Commun Biol. 2023 Feb 21. 6(1): 205

A non-canonical sensing pathway mediates Plasmodium adaptation to amino acid deficiency.

Inês M Marreiros, Sofia Marques, Ana Parreira, Vincent Mastrodomenico, Bryan C Mounce, Chantal T Harris, Björn F Kafsack, Oliver Billker, Vanessa Zuzarte-Luís, Maria M Mota.

Eukaryotes have canonical pathways for responding to amino acid (AA) availability. Under AA-limiting conditions, the TOR complex is repressed, whereas the sensor kinase GCN2 is activated. While these pathways have been highly conserved throughout evolution, malaria parasites are a rare exception. Despite auxotrophic for most AA, Plasmodium does not have either a TOR complex nor the GCN2-downstream transcription factors. While Ile starvation has been shown to trigger eIF2α phosphorylation and a hibernation-like response, the overall mechanisms mediating detection and response to AA fluctuation in the absence of such pathways has remained elusive. Here we show that Plasmodium parasites rely on an efficient sensing pathway to respond to AA fluctuations. A phenotypic screen of kinase knockout mutant parasites identified nek4, eIK1 and eIK2-the last two clustering with the eukaryotic eIF2α kinases-as critical for Plasmodium to sense and respond to distinct AA-limiting conditions. Such AA-sensing pathway is temporally regulated at distinct life cycle stages, allowing parasites to actively fine-tune replication and development in response to AA availability. Collectively, our data disclose a set of heterogeneous responses to AA depletion in malaria parasites, mediated by a complex mechanism that is critical for modulating parasite growth and survival.

DOI: https://doi.org/10.1038/s42003-023-04566-y
Autophagy. 2023 Feb 20.

Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.

Yongook Lee, Bongkeun Kim, Hae-Soo Jang, Won-Ki Huh.

  Macroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS.

Keywords:  Atg1; Atg18; Atg8; Saccharomyces cerevisiae; Vps34; autophagy; nitrogen starvation

DOI:  https://doi.org/10.1080/15548627.2023.2182478
PeerJ. 2023 ;11 e14752

Implications of altered sirtuins in metabolic regulation and oral cancer.

Xu Quan, Ying Xin, He-Ling Wang, Yingjie Sun, Chanchan Chen, Jiangying Zhang.

  Sirtuins (SIRTs 1-7) are a group of histone deacetylase enzymes with a wide range of enzyme activities that target a range of cellular proteins in the nucleus, cytoplasm, and mitochondria for posttranslational modifications by acetylation (SIRT1, 2, 3, and 5) or ADP ribosylation (SIRT4, 6, and 7). A variety of cellular functions, including mitochondrial functions and functions in energy homeostasis, metabolism, cancer, longevity and ageing, are regulated by sirtuins. Compromised sirtuin functions and/or alterations in the expression levels of sirtuins may lead to several pathological conditions and contribute significantly to alterations in metabolic phenotypes as well as oral carcinogenesis. Here, we describe the basic characteristics of seven mammalian sirtuins. This review also emphasizes the key molecular mechanisms of sirtuins in metabolic regulation and discusses the possible relationships of sirtuins with oral cancers. This review will provide novel insight into new therapeutic approaches targeting sirtuins that may potentially lead to effective strategies for combating oral malignancies.

Keywords:  Metabolic regulation; Oral cancer; Sirtuin

DOI:  https://doi.org/10.7717/peerj.14752