bims-toxgon 2023-07-09 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–07–09
eleven papers selected by
Lakesh Kumar, BITS Pilani

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.
Toxoplasma gondii encodes an array of TBC-domain containing proteins including an essential regulator that targets Rab2 in the secretory pathway.
The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.
Repurposing the Medicines for Malaria Venture's COVID Box to discover potent inhibitors of Toxoplasma gondii, and in vivo efficacy evaluation of almitrine bismesylate (MMV1804175) in chronically infected mice.
Unique Properties of Apicomplexan Mitochondria.
A PPP-type pseudophosphatase is required for the maintenance of basal complex integrity in Plasmodium falciparum.
Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.
Multifaceted functions of histone deacetylases in stress response.
ATP-citrate lyase controls endothelial gluco-lipogenic metabolism and vascular inflammation in sepsis-associated organ injury.
The role of Vps4 in cancer development.
The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.

bioRxiv. 2023 Jun 13. pii: 2023.06.13.544803. [Epub ahead of print]

The protein phosphatase PPKL is a key regulator of daughter parasite development in Toxoplasma gondii.

Chunlin Yang, Emma H Doud, Emily Sampson, Gustavo Arrizabalaga.

Apicomplexan parasites, including Toxoplasma gondii , encode many plant-like proteins, which play significant roles and present attractive targets for drug development. In this study, we have characterized the plant-like protein phosphatase PPKL, which is unique to the parasite and absent in its mammalian host. We have shown that its localization changes as the parasite divides. In non-dividing parasites, it is present in the cytoplasm, nucleus, and preconoidal region. As the parasite begins division, PPKL is enriched in the preconoidal region and the cortical cytoskeleton of the nascent parasites. Later in the division, PPKL is present in the basal complex ring. Conditional knockdown of PPKL showed that it is essential for parasite propagation. Moreover, parasites lacking PPKL exhibit uncoupling of division, with normal DNA duplication but severe defects in forming daughter parasites. While PPKL depletion does not impair the duplication of centrosomes, it affects the rigidity and arrangement of the cortical microtubules. Both Co-Immunoprecipitation and proximity labeling identified the kinase DYRK1 as a potential functional partner of PPKL. Complete knockout of DYRK1 phenocopies lack of PPKL, strongly suggesting a functional relationship between these two signaling proteins. Global phosphoproteomics analysis revealed a significant increase in phosphorylation of the microtubule-associated proteins SPM1 in PPKL-depleted parasites, suggesting PPKL regulates the cortical microtubules by mediating the phosphorylation state of SPM1. More importantly, the phosphorylation of cell cycle-associated kinase Crk1, a known regulator of daughter cell assembly, is altered in PPKL-depleted parasites. Thus, we propose that PPKL regulates daughter parasite development by influencing the Crk1-dependent signaling pathway.
Importance: Toxoplasma gondii can cause severe disease in immunocompromised or immunosuppressed patients and during congenital infections. Treating toxoplasmosis presents enormous challenges since the parasite shares many biological processes with its mammalian hosts, which results in significant side effects with current therapies. Consequently, proteins that are essential and unique to the parasite represent favorable targets for drug development. Interestingly, Toxoplasma , like other members of the phylum Apicomplexa, has numerous plant-like proteins, many of which play crucial roles and do not have equivalents in the mammalian host. In this study, we found that the plant-like protein phosphatase, PPKL, appears to be a key regulator of daughter parasite development. With the depletion of PPKL, the parasite shows severe defects in forming daughter parasites. This study provides novel insights into the understanding of parasite division and offers a new potential target for the development of antiparasitic drugs.

DOI: https://doi.org/10.1101/2023.06.13.544803
bioRxiv. 2023 May 29. pii: 2023.05.28.542599. [Epub ahead of print]

Toxoplasma gondii encodes an array of TBC-domain containing proteins including an essential regulator that targets Rab2 in the secretory pathway.

Justin J Quan, Lachezar A Nikolov, Jihui Sha, James A Wohlschlegel, Peter J Bradley.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host-cell manipulation and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii , precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC)-domain containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC-domain containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. We then use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein that localizes to the ER is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and Golgi apparatus. We show that the conserved dual-finger active site in the TBC-domain of the protein is critical for its GTPase-activating protein (GAP) function and that the P. falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast two hybrid analyses that TgTBC9 directly binds Rab2, indicating that this TBC-Rab pair controls ER to Golgi traffic in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan, provide new insight into intracellular vesicle trafficking in T. gondii , and reveal promising targets for the design of novel therapeutics that can specifically target apicomplexan parasites.

DOI: https://doi.org/10.1101/2023.05.28.542599
bioRxiv. 2023 Jun 01. pii: 2023.05.31.543158. [Epub ahead of print]

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Amara C Thind, Caroline M Mota, Ana Paula N Gonçalves, Jihui Sha, James A Wohlschlegel, Tiago W P Mineo, Peter J Bradley.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden.
Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

DOI: https://doi.org/10.1101/2023.05.31.543158
PLoS One. 2023 ;18(7): e0288335

Repurposing the Medicines for Malaria Venture's COVID Box to discover potent inhibitors of Toxoplasma gondii, and in vivo efficacy evaluation of almitrine bismesylate (MMV1804175) in chronically infected mice.

Bruna Ramos Dos Santos, Amanda Bruno da Silva Bellini Ramos, Renata Priscila Barros de Menezes, Marcus Tullius Scotti, Fábio Antônio Colombo, Marcos José Marques, Juliana Quero Reimão.

Toxoplasmosis, caused by the obligate intracellular parasite Toxoplasma gondii, affects about one-third of the world's population and can cause severe congenital, neurological and ocular issues. Current treatment options are limited, and there are no human vaccines available to prevent transmission. Drug repurposing has been effective in identifying anti-T. gondii drugs. In this study, the screening of the COVID Box, a compilation of 160 compounds provided by the "Medicines for Malaria Venture" organization, was conducted to explore its potential for repurposing drugs to combat toxoplasmosis. The objective of the present work was to evaluate the compounds' ability to inhibit T. gondii tachyzoite growth, assess their cytotoxicity against human cells, examine their absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties, and investigate the potential of one candidate drug through an experimental chronic model of toxoplasmosis. Early screening identified 29 compounds that could inhibit T. gondii survival by over 80% while keeping human cell survival up to 50% at a concentration of 1 μM. The Half Effective Concentrations (EC50) of these compounds ranged from 0.04 to 0.92 μM, while the Half Cytotoxic Concentrations (CC50) ranged from 2.48 to over 50 μM. Almitrine was chosen for further evaluation due to its favorable characteristics, including anti-T. gondii activity at nanomolar concentrations, low cytotoxicity, and ADMET properties. Administering almitrine bismesylate (Vectarion®) orally at dose of 25 mg/kg/day for ten consecutive days resulted in a statistically significant (p < 0.001) reduction in parasite burden in the brains of mice chronically infected with T. gondii (ME49 strain). This was determined by quantifying the RNA of living parasites using real-time PCR. The presented results suggest that almitrine may be a promising drug candidate for additional experimental studies on toxoplasmosis and provide further evidence of the potential of the MMV collections as a valuable source of drugs to be repositioned for infectious diseases.

DOI: https://doi.org/10.1371/journal.pone.0288335
Annu Rev Microbiol. 2023 Jul 05.

Unique Properties of Apicomplexan Mitochondria.

Ian M Lamb, Ijeoma C Okoye, Michael W Mather, Akhil B Vaidya.

Apicomplexan parasites constitute more than 6,000 species infecting a wide range of hosts. These include important pathogens such as those causing malaria and toxoplasmosis. Their evolutionary emergence coincided with the dawn of animals. Mitochondrial genomes of apicomplexan parasites have undergone dramatic reduction in their coding capacity, with genes for only three proteins and ribosomal RNA genes present in scrambled fragments originating from both strands. Different branches of the apicomplexans have undergone rearrangements of these genes, with Toxoplasma having massive variations in gene arrangements spread over multiple copies. The vast evolutionary distance between the parasite and the host mitochondria has been exploited for the development of antiparasitic drugs, especially those used to treat malaria, wherein inhibition of the parasite mitochondrial respiratory chain is selectively targeted with little toxicity to the host mitochondria. We describe additional unique characteristics of the parasite mitochondria that are being investigated and provide greater insights into these deep-branching eukaryotic pathogens. Expected final online publication date for the Annual Review of Microbiology, Volume 77 is September 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

DOI: https://doi.org/10.1146/annurev-micro-032421-120540
Nat Commun. 2023 07 03. 14(1): 3916

A PPP-type pseudophosphatase is required for the maintenance of basal complex integrity in Plasmodium falciparum.

Alexander A Morano, Rachel M Rudlaff, Jeffrey D Dvorin.

During its asexual blood stage, P. falciparum replicates via schizogony, wherein dozens of daughter cells are formed within a single parent. The basal complex, a contractile ring that separates daughter cells, is critical for schizogony. In this study, we identify a Plasmodium basal complex protein essential for basal complex maintenance. Using multiple microscopy techniques, we demonstrate that PfPPP8 is required for uniform basal complex expansion and maintenance of its integrity. We characterize PfPPP8 as the founding member of a novel family of pseudophosphatases with homologs in other Apicomplexan parasites. By co-immunoprecipitation, we identify two additional new basal complex proteins. We characterize the unique temporal localizations of these new basal complex proteins (late-arriving) and of PfPPP8 (early-departing). In this work, we identify a novel basal complex protein, determine its specific role in segmentation, identify a new pseudophosphatase family, and establish that the P. falciparum basal complex is a dynamic structure.

DOI: https://doi.org/10.1038/s41467-023-39435-z
bioRxiv. 2023 Jun 01. pii: 2023.06.01.543311. [Epub ahead of print]

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A.

Daniel S Kantner, Anna Bostwick, Vicky Yang, Emily Megill, Carmen Bekeova, Erin Seifert, Nathaniel W Snyder.

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards.

DOI: https://doi.org/10.1101/2023.06.01.543311
Trends Plant Sci. 2023 Jul 01. pii: S1360-1385(23)00198-X. [Epub ahead of print]

Multifaceted functions of histone deacetylases in stress response.

Xiaoyun Cui, Avilien Dard, Jean-Philippe Reichheld, Dao-Xiu Zhou.

  Histone deacetylases (HDACs) are important chromatin regulators essential for plant tolerance to adverse environments. In addition to histone deacetylation and epigenetic regulation, HDACs deacetylate non-histone proteins and thereby regulate multiple pathways. Like other post-translational modifications (PTMs), acetylation/deacetylation is a reversible switch regulating different cellular processes in plants. Here, by focusing on results obtained in arabidopsis (Arabidopsis thaliana) and rice plants, we analyze the different aspects of HDAC functions and the underlying regulatory mechanisms in modulating plant responses to stress. We hypothesize that, in addition to epigenetic regulation of gene expression, HDACs can also control plant tolerance to stress by regulating transcription, translation, and metabolic activities and possibly assembly-disassembly of stress granules (SGs) through lysine deacetylation of non-histone proteins.

Keywords:  histone deacetylase; metabolic enzymes; protein lysine acetylation/acylation; stress granules; transcription factors

DOI:  https://doi.org/10.1016/j.tplants.2023.06.006
Cell Death Dis. 2023 Jul 06. 14(7): 401

ATP-citrate lyase controls endothelial gluco-lipogenic metabolism and vascular inflammation in sepsis-associated organ injury.

Ranran Li, Mei Meng, Ying Chen, Tingting Pan, Yinjiaozhi Li, Yunxin Deng, Ruyuan Zhang, Rui Tian, Wen Xu, Xiangtao Zheng, Fangchen Gong, Jie Liu, Haiting Tang, Xiaowei Ding, Yaoqing Tang, Djillali Annane, Erzhen Chen, Hongping Qu, Lei Li.

Sepsis involves endothelial cell (EC) dysfunction, which contributes to multiple organ failure. To improve therapeutic prospects, elucidating molecular mechanisms of vascular dysfunction is of the essence. ATP-citrate lyase (ACLY) directs glucose metabolic fluxes to de novo lipogenesis by generating acetyl-Co-enzyme A (acetyl-CoA), which facilitates transcriptional priming via protein acetylation. It is well illustrated that ACLY participates in promoting cancer metastasis and fatty liver diseases. Its biological functions in ECs during sepsis remain unclear. We found that plasma levels of ACLY were increased in septic patients and were positively correlated with interleukin (IL)-6, soluble E-selectin (sE-selectin), soluble vascular cell adhesion molecule 1 (sVCAM-1), and lactate levels. ACLY inhibition significantly ameliorated lipopolysaccharide challenge-induced EC proinflammatory response in vitro and organ injury in vivo. The metabolomic analysis revealed that ACLY blockade fostered ECs a quiescent status by reducing the levels of glycolytic and lipogenic metabolites. Mechanistically, ACLY promoted forkhead box O1 (FoxO1) and histone H3 acetylation, thereby increasing the transcription of c-Myc (MYC) to facilitate the expression of proinflammatory and gluco-lipogenic genes. Our findings revealed that ACLY promoted EC gluco-lipogenic metabolism and proinflammatory response through acetylation-mediated MYC transcription, suggesting ACLY as the potential therapeutic target for treating sepsis-associated EC dysfunction and organ injury.

DOI: https://doi.org/10.1038/s41419-023-05932-8
Front Oncol. 2023 ;13 1203359

The role of Vps4 in cancer development.

Li Juan Huang, Shi Tong Zhan, Yu Qin Pan, Wei Bao, Ye Yang.

  VPS4 series proteins play a crucial role in the endosomal sorting complexes required for the transport (ESCRT) pathway, which is responsible for sorting and trafficking cellular proteins and is involved in various cellular processes, including cytokinesis, membrane repair, and viral budding. VPS4 proteins are ATPases that mediate the final steps of membrane fission and protein sorting as part of the ESCRT machinery. They disassemble ESCRT-III filaments, which are vital for forming multivesicular bodies (MVBs) and the release of intraluminal vesicles (ILVs), ultimately leading to the sorting and degradation of various cellular proteins, including those involved in cancer development and progression. Recent studies have shown a potential relationship between VPS4 series proteins and cancer. Evidence suggests that these proteins may have crucial roles in cancer development and progression. Several experiments have explored the association between VPS4 and different types of cancer, including gastrointestinal and reproductive system tumors, providing insight into the underlying mechanisms. Understanding the structure and function of VPS4 series proteins is critical in assessing their potential role in cancer. The evidence supporting the involvement of VPS4 series proteins in cancer provides a promising avenue for future research and therapeutic development. However, further researches are necessary to fully understand the mechanisms underlying the relationship between VPS4 series proteins and cancer and to develop effective strategies for targeting these proteins in cancer therapy. This article aims to review the structures and functions of VPS4 series proteins and the previous experiments to analyze the relationship between VPS4 series proteins and cancer.

Keywords:  Vps4; cancer; cell death; exosome; mechanisms

DOI:  https://doi.org/10.3389/fonc.2023.1203359
Front Mol Biosci. 2023 ;10 1168680

The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.

Farzana Tuli, Patricia M Kane.

  Vacuolar H+-ATPases (V-ATPases) acidify several organelles in all eukaryotic cells and export protons across the plasma membrane in a subset of cell types. V-ATPases are multisubunit enzymes consisting of a peripheral subcomplex, V1, that is exposed to the cytosol and an integral membrane subcomplex, Vo, that contains the proton pore. The Vo a-subunit is the largest membrane subunit and consists of two domains. The N-terminal domain of the a-subunit (aNT) interacts with several V1 and Vo subunits and serves to bridge the V1 and Vo subcomplexes, while the C-terminal domain contains eight transmembrane helices, two of which are directly involved in proton transport. Although there can be multiple isoforms of several V-ATPase subunits, the a-subunit is encoded by the largest number of isoforms in most organisms. For example, the human genome encodes four a-subunit isoforms that exhibit a tissue- and organelle-specific distribution. In the yeast S. cerevisiae, the two a-subunit isoforms, Golgi-enriched Stv1 and vacuolar Vph1, are the only V-ATPase subunit isoforms. Current structural information indicates that a-subunit isoforms adopt a similar backbone structure but sequence variations allow for specific interactions during trafficking and in response to cellular signals. V-ATPases are subject to several types of environmental regulation that serve to tune their activity to their cellular location and environmental demands. The position of the aNT domain in the complex makes it an ideal target for modulating V1-Vo interactions and regulating enzyme activity. The yeast a-subunit isoforms have served as a paradigm for dissecting interactions of regulatory inputs with subunit isoforms. Importantly, structures of yeast V-ATPases containing each a-subunit isoform are available. Chimeric a-subunits combining elements of Stv1NT and Vph1NT have provided insights into how regulatory inputs can be integrated to allow V-ATPases to support cell growth under different stress conditions. Although the function and distribution of the four mammalian a-subunit isoforms present additional complexity, it is clear that the aNT domains of these isoforms are also subject to multiple regulatory interactions. Regulatory mechanisms that target mammalian a-subunit isoforms, and specifically the aNT domains, will be described. Altered V-ATPase function is associated with multiple diseases in humans. The possibility of regulating V-ATPase subpopulations via their isoform-specific regulatory interactions are discussed.

Keywords:  V-ATPase; a-subunit; acidification; isoform; organelle; regulation; reversible disassembly

DOI:  https://doi.org/10.3389/fmolb.2023.1168680