bims-toxgon 2023-05-14 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–05–14
eleven papers selected by
Lakesh Kumar, BITS Pilani

Mitochondrial ADP/ATP Carrier 1 Is Important for the Growth of Toxoplasma Tachyzoites.
MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.
The cortical microtubules of Toxoplasma contribute to the helicity, but not the speed or persistence of parasite movement.
Key substrate recognition residues in the active site of cystathionine beta-synthase from Toxoplasma gondii.
Shedding light on a mysterious link between Toxoplasma gondii and cancer: A review.
The Plasmodium falciparum apicoplast cysteine desulfurase provides sulfur for both iron sulfur cluster assembly and tRNA modification.
Examination of Secondary Metabolite Biosynthesis in Apicomplexa.
Cryptosporidium secretome sheds new light on host-parasite interactions.
Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors.
Encystation stimuli sensing mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia.
Vps13 is required for efficient autophagy in Saccharomyces cerevisiae.

Microbiol Spectr. 2023 May 08. e0004023

Mitochondrial ADP/ATP Carrier 1 Is Important for the Growth of Toxoplasma Tachyzoites.

Jiahui Qian, Tongjie Zhao, Liyu Guo, Senyang Li, Zhengming He, Mingfeng He, Bang Shen, Rui Fang.

  Metabolism associated with energy production is highly compartmentalized in eukaryotic cells. During this process, transporters that move metabolites across organelle membranes play pivotal roles. The highly conserved ADP/ATP carrier (AAC) involved in ATP and ADP exchange between the mitochondria and cytoplasm is key to linking the metabolic activities in these 2 compartments. The ATP produced in mitochondria can be exchanged with cytoplasmic ADP by AAC, thus satisfying the energy needs in the cytoplasm. Toxoplasma gondii is an obligate intracellular parasite with a wide range of hosts. Previous studies have shown that mitochondrial metabolism helps Toxoplasma to parasitize diverse host cells. Here, we identified 2 putative mitochondria ADP/ATP carriers in Toxoplasma with significant sequence similarity to known AACs from other eukaryotes. We examined the ATP transport function of TgAACs by expressing them in Escherichia coli cells and found that only TgAAC1 had ATP transport activity. Moreover, knockdown of TgAAC1 caused severe growth defects of parasites and heterologous expression of mouse ANT2 in the TgAAC1 depletion mutant restored its growth, revealing its importance for parasite growth. These results verified that TgAAC1 functions as the mitochondrial ADP/ATP carrier in T. gondii and the functional studies demonstrated the importance of TgAAC1 for tachyzoites growth. IMPORTANCE T. gondii has an efficient and flexible energy metabolism system to meet different growth needs. ATP is an energy-carrying molecule and needs to be exchanged between organelles with the assistance of transporters. However, the function of TgAACs has yet to be characterized. Here, we identified 2 putative AACs of T. gondii and verified that only TgAAC1 had ATP transport activity with expression in the intact E. coli cells. Detailed analyses found that TgAAC1 is critical for the growth of tachyzoites and TgAAC2 is dispensable. Moreover, complementation with mouse ANT2 restored the growth speed of iTgAAC1, further suggesting TgAAC1 functions as a mitochondrial ADP/ATP carrier. Our research demonstrated the importance of TgAAC1 for tachyzoites growth.

Keywords:  ATP; TgAAC1; Toxoplasma gondii; mitochondrial ADP/ATP carrier

DOI:  https://doi.org/10.1128/spectrum.00040-23
PLoS Biol. 2023 May 08. 21(5): e3002110

MyosinA is a druggable target in the widespread protozoan parasite Toxoplasma gondii.

Anne Kelsen, Robyn S Kent, Anne K Snyder, Eddie Wehri, Stephen J Bishop, Rachel V Stadler, Cameron Powell, Bruno Martorelli di Genova, Pramod K Rompikuntal, Martin J Boulanger, David M Warshaw, Nicholas J Westwood, Julia Schaletzky, Gary E Ward.

Toxoplasma gondii is a widespread apicomplexan parasite that can cause severe disease in its human hosts. The ability of T. gondii and other apicomplexan parasites to invade into, egress from, and move between cells of the hosts they infect is critical to parasite virulence and disease progression. An unusual and highly conserved parasite myosin motor (TgMyoA) plays a central role in T. gondii motility. The goal of this work was to determine whether the parasite's motility and lytic cycle can be disrupted through pharmacological inhibition of TgMyoA, as an approach to altering disease progression in vivo. To this end, we first sought to identify inhibitors of TgMyoA by screening a collection of 50,000 structurally diverse small molecules for inhibitors of the recombinant motor's actin-activated ATPase activity. The top hit to emerge from the screen, KNX-002, inhibited TgMyoA with little to no effect on any of the vertebrate myosins tested. KNX-002 was also active against parasites, inhibiting parasite motility and growth in culture in a dose-dependent manner. We used chemical mutagenesis, selection in KNX-002, and targeted sequencing to identify a mutation in TgMyoA (T130A) that renders the recombinant motor less sensitive to compound. Compared to wild-type parasites, parasites expressing the T130A mutation showed reduced sensitivity to KNX-002 in motility and growth assays, confirming TgMyoA as a biologically relevant target of KNX-002. Finally, we present evidence that KNX-002 can slow disease progression in mice infected with wild-type parasites, but not parasites expressing the resistance-conferring TgMyoA T130A mutation. Taken together, these data demonstrate the specificity of KNX-002 for TgMyoA, both in vitro and in vivo, and validate TgMyoA as a druggable target in infections with T. gondii. Since TgMyoA is essential for virulence, conserved in apicomplexan parasites, and distinctly different from the myosins found in humans, pharmacological inhibition of MyoA offers a promising new approach to treating the devastating diseases caused by T. gondii and other apicomplexan parasites.

DOI: https://doi.org/10.1371/journal.pbio.3002110
bioRxiv. 2023 Apr 25. pii: 2023.04.23.538011. [Epub ahead of print]

The cortical microtubules of Toxoplasma contribute to the helicity, but not the speed or persistence of parasite movement.

Isadonna F Tengganu, Luisa F Arias Padilla, Jonathan Munera Lopez, Jun Liu, Peter T Brown, Ke Hu.

Apicomplexans parasitize a wide range of hosts. To infect, the parasite needs to travel through different types of tissues and invade into various types of cells. In tissues and three-dimensional (3-D) matrix, the apicomplexan parasite Toxoplasma gondii moves along a helical path. While many genes, including actin and myosins, have been shown to be important for parasite motility, it remains unknown what allows the parasite to travel over a long distance along a helical path. The cortical microtubules, which are ultra-stable, sprially arranged, and form extensive lateral interaction with the parasite cortex, have been considered to be a candidate structure for guiding the long-distance movement of the parasite. In wild-type parasites, the cortical microtubules in mature parasites are impervious to destabilization by cold-treatment or depolymerizing drugs, which makes it difficult to assess their function. Previously, we generated a mutant (dubbed "TKO" for succinctness) that lacks three microtubule-associated proteins. The loss of these three proteins destabilizes the cortical microtubules in mature parasites. Here we quantify the proportion of parasites with various levels of defects in the microtubule array under different conditions. We found that ∽ 80% of the non-dividing TKO parasites have severely curtailed cortical microtubules. The extent of depolymerization is further exacerbated upon the initiation of daughter construction or cold treatment. The cold-induced depolymerization is reversible, with noticeable restoration of the cortical microtubules within 20 min of temperature shift to 37°C. While microtubule polymerization is essential for generating viable daughter cells, the destabilization of the cortical microtubules in the mature parasite does not affect parasite replication. In a 3-D Matrigel matrix, the TKO mutant parasites can travel directionally over long distances. However, their trajectories are significantly more linear than those of wild-type parasites. In tissue culture, the TKO parasite displays a defect in infection and cytolytic efficiency. Interestingly, the speed and behavior of the parasite's entry into and egress from the host cell are similar to that of the wild-type parasite. These results indicate that the cortical microtubules contribute to the helicity but not the persistence of parasite long-distance movement. Furthermore, host cell entry is less sensitive to structural changes in the parasite than overall infection efficiency, which also include extracellular migration and orient the parasite for proper contact with the host cell.

DOI: https://doi.org/10.1101/2023.04.23.538011
Proteins. 2023 May 10.

Key substrate recognition residues in the active site of cystathionine beta-synthase from Toxoplasma gondii.

Carolina Conter, Filippo Favretto, Paola Dominici, Luis Alfonso Martinez-Cruz, Alessandra Astegno.

  Cystathionine β-synthase (CBS) catalyzes the condensation of l-serine and l-homocysteine to give l-cystathionine in the transsulfuration pathway. Recently, a few O-acetylserine (l-OAS)-dependent CBSs (OCBSs) have been found in bacteria that can exclusively function with l-OAS. CBS from Toxoplasma gondii (TgCBS) can efficiently use both l-serine and l-OAS to form l-cystathionine. In this work, a series of site-specific variants substituting S84, Y160, and Y246 with hydrophobic residues found at the same positions in OCBSs was generated to explore the roles of the hydroxyl moieties of these residues as determinants of l-serine/l-OAS preference in TgCBS. We found that the S84A/Y160F/Y246V triple mutant behaved like an OCBS in terms of both substrate requirements, showing β-replacement activity only with l-OAS, and pH optimum, which is decreased by ~1 pH unit. Formation of a stable aminoacrylate upon reaction with l-serine is prevented by the triple mutation, indicating the importance of the H-bonds between the hydroxyl groups of Y160, Y246, and S84 with l-serine in formation of the intermediate. Analysis of the independent effect of each mutation on TgCBS activity and investigation of the protein-aminoacrylate complex structure allowed for the conclusion that the hydroxyl group of Y246 has a major, but not exclusive, role in controlling the l-serine preference by efficiently stabilizing its leaving group. These studies demonstrate that differences in substrate specificity of CBSs are controlled by natural variations in as few as three residue positions. A better understanding of substrate specificity in TgCBS will facilitate the design of new antimicrobial compounds.

Keywords:  Toxoplasma gondii; cystathionine β-synthase; enzyme kinetics; mutagenesis; substrate specificity

DOI:  https://doi.org/10.1002/prot.26507
Exp Parasitol. 2023 May 05. pii: S0014-4894(23)00085-1. [Epub ahead of print]250 108544

Shedding light on a mysterious link between Toxoplasma gondii and cancer: A review.

Nahla El Skhawy, Maha M Eissa.

  The ongoing conflict regarding the affiliation of Toxoplasma gondii to cancer; whether an inducer or a suppressor needs to be resolved. Human epidemiological studies oscillate without attaining a firm ground. Some studies confirmed the detection of high seroprevalence of anti-Toxoplasma antibodies in different cancer patients without further justification whether being causation, co-incidences, or part of opportunistic infections. Others reported a state of resistance to cancer accompanying low titer of anti-Toxoplasma antibody. Worthwhile, preclinical experimental work confirmed the antineoplastic potency of Toxoplasma. Thus, further investigational research is essential to validate the potential application of Toxoplasma as a promising cancer immunotherapeutic vaccine candidate. In this paper, we present a review of this issue by examining epidemiological and preclinical experimental studies that explored the linkage between Toxoplasma gondii and cancer. We consider this review an important step towards shedding a light on this mysterious link and a stepping-stone for potential research work addressing Toxoplasma as a cancer suppressor rather than a cancer inducer.

Keywords:  Cancer; Cancer promoters; Cancer suppressor; Pathogens and cancer; Toxoplasma gondii

DOI:  https://doi.org/10.1016/j.exppara.2023.108544
Elife. 2023 May 11. pii: e84491. [Epub ahead of print]12

The Plasmodium falciparum apicoplast cysteine desulfurase provides sulfur for both iron sulfur cluster assembly and tRNA modification.

Russell P Swift, Rubayet Elahi, Krithika Rajaram, Hans B Liu, Sean T Prigge.

  Iron sulfur clusters (FeS) are ancient and ubiquitous protein cofactors that play fundamental roles in many aspects of cell biology. These cofactors cannot be scavenged or trafficked within a cell and thus must be synthesized in any subcellular compartment where they are required. We examined the FeS synthesis proteins found in the relict plastid organelle, called the apicoplast, of the human malaria parasite Plasmodium falciparum. Using a chemical bypass method, we deleted four of the FeS pathway proteins involved in sulfur acquisition and cluster assembly and demonstrated that they are all essential for parasite survival. However, the effect that these deletions had on the apicoplast organelle differed. Deletion of the cysteine desulfurase SufS led to disruption of the apicoplast organelle and loss of the organellar genome, whereas the other deletions did not affect organelle maintenance. Ultimately, we discovered that the requirement of SufS for organelle maintenance is not driven by its role in FeS biosynthesis, but rather, by its function in generating sulfur for use by MnmA, a tRNA modifying enzyme that we localized to the apicoplast. Complementation of MnmA and SufS activity with a bacterial MnmA and its cognate cysteine desulfurase, strongly suggests that the parasite SufS provides sulfur for both FeS biosynthesis and tRNA modification in the apicoplast. The dual role of parasite SufS is likely to be found in other plastid-containing organisms and highlights the central role of this enzyme in plastid biology.

Keywords:  P. falciparum; infectious disease; microbiology

DOI:  https://doi.org/10.7554/eLife.84491
Chembiochem. 2023 May 12. e202300263

Examination of Secondary Metabolite Biosynthesis in Apicomplexa.

Hannah D'Ambrosio, Aaron Keeler, Emily Derbyshire.

  Natural product discovery has traditionally relied on the isolation of small molecules from producing species, but genome sequencing technology and advances in molecular biology techniques have expanded efforts to a wider array of organisms. Protists represent an underexplored kingdom for specialized metabolite searches despite bioinformatic analysis that suggests they harbor distinct biologically active small molecules. Specifically, pathogenic apicomplexan parasites, responsible for billions of global infections, have been found to possess multiple biosynthetic gene clusters, which hints at their capacity to produce polyketide metabolites. Biochemical studies have revealed unique features of apicomplexan polyketide synthases, but to date, the identity and function of polyketides synthesized by these megaenzymes remains unknown. Herein, we discuss the potential for specialized metabolite production in protists and the possible evolution of polyketide biosynthetic gene clusters in apicomplexan parasites. We then focus on a polyketide synthase from the apicomplexan Toxoplasma gondii to discuss the unique domain architecture and properties of these proteins when compared to previously characterized systems, and further speculate on the possible functions for polyketides in these pathogenic parasites.

Keywords:  Apicomplexa; Polyketide Synthase; Toxoplasma gondii; biosynthesis; natural products

DOI:  https://doi.org/10.1002/cbic.202300263
Trends Parasitol. 2023 May 05. pii: S1471-4922(23)00100-9. [Epub ahead of print]

Cryptosporidium secretome sheds new light on host-parasite interactions.

Isabelle Coppens, Honorine D Ward.

  Invasive Cryptosporidium sporozoites contain organelles that secrete unique proteins to facilitate invasion and remodeling of the infected cell. By identifying a novel secretory organelle, 'small granules', and defining the global content of all the secretory organelles, Guérin et al. set the stage to uncover molecular determinants of virulence at the host cell interface.

Keywords:  Cryptosporidium; host–parasite interactions; proteome; secretory organelles

DOI:  https://doi.org/10.1016/j.pt.2023.04.008
bioRxiv. 2023 Apr 28. pii: 2023.04.24.538118. [Epub ahead of print]

Metabolic Reprogramming by Histone Deacetylase Inhibition Selectively Targets NRF2-activated tumors.

Dimitris Karagiannis, Warren Wu, Albert Li, Makiko Hayashi, Xiao Chen, Michaela Yip, Vaibhav Mangipudy, Xinjing Xu, Francisco J Sánchez-Rivera, Yadira M Soto-Feliciano, Jiangbin Ye, Thales Papagiannakopoulos, Chao Lu.

Interplay between metabolism and chromatin signaling have been implicated in cancer initiation and progression. However, whether and how metabolic reprogramming in tumors generates specific epigenetic vulnerabilities remain unclear. Lung adenocarcinoma (LUAD) tumors frequently harbor mutations that cause aberrant activation of the NRF2 antioxidant pathway and drive aggressive and chemo-resistant disease. We performed a chromatin-focused CRISPR screen and report that NRF2 activation sensitized LUAD cells to genetic and chemical inhibition of class I histone deacetylases (HDAC). This association was consistently observed across cultured cells, syngeneic mouse models and patient-derived xenografts. HDAC inhibition causes widespread increases in histone H4 acetylation (H4ac) at intergenic regions, but also drives re-targeting of H4ac reader protein BRD4 away from promoters with high H4ac levels and transcriptional downregulation of corresponding genes. Integrative epigenomic, transcriptomic and metabolomic analysis demonstrates that these chromatin changes are associated with reduced flux into amino acid metabolism and de novo nucleotide synthesis pathways that are preferentially required for the survival of NRF2-active cancer cells. Together, our findings suggest that metabolic alterations such as NRF2 activation could serve as biomarkers for effective repurposing of HDAC inhibitors to treat solid tumors.

DOI: https://doi.org/10.1101/2023.04.24.538118
Res Sq. 2023 Apr 25. pii: rs.3.rs-2818450. [Epub ahead of print]

Encystation stimuli sensing mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia.

Alexander Paredez, Han-Wei Shih, Germain Alas.

Protozoan parasites use cAMP signaling to precisely regulate the place and time of developmental differentiation, yet it is unclear how this signaling is initiated. Encystation of the intestinal parasite Giardia lamblia can be activated by multiple stimuli, which we hypothesize result in a common physiological change. We demonstrate that bile alters plasma membrane fluidity by reducing cholesterol-rich lipid microdomains, while alkaline pH enhances bile function. Through depletion of the cAMP producing enzyme Adenylate Cyclase 2 (AC2) and the use of a newly developed Giardia-specific cAMP sensor, we show that AC2 is necessary for encystation stimuli-induced cAMP upregulation and activation of downstream signaling. Conversely, over expression of AC2 or exogenous cAMP were sufficient to initiate encystation. Our findings indicate that encystation stimuli induce membrane reorganization, trigger AC2-dependent cAMP upregulation, and initiate encystation-specific gene expression, thereby advancing our understanding of a critical stage in the life cycle of a globally important parasite.

DOI: https://doi.org/10.21203/rs.3.rs-2818450/v1
Contact (Thousand Oaks). 2022 Jan-Dec;5:5

Vps13 is required for efficient autophagy in Saccharomyces cerevisiae.

Yuchen Lei, Xin Wen, Daniel J Klionsky.

  Vps13 is a large, conserved protein that transports lipids between membranes. Its localization at multiple organelle membranes and membrane contact sites suggests its important physiological roles. In addition, the high correlation of mutant VPS13 with certain diseases, especially those involving neurodegeneration, makes this protein of considerable biomedical interest. Taking advantage of the fact that yeasts only have one Vps13 protein, the roles of yeast Vps13 have been well studied. However, whether and how Vps13 functions in macroautophagy/autophagy, a process of degradation of cytoplasmic cargoes, have been elusive questions. In this paper, we investigated the role of Vps13 in both non-selective and selective autophagy and found that this protein participates in non-selective autophagy, reticulophagy and pexophagy, but not mitophagy, and that Vps13 plays a role in the late stage of autophagy.

Keywords:  Membrane; mitophagy; protein trafficking; stress; vacuole

DOI:  https://doi.org/10.1177/25152564221136388