bims-toxgon 2023-01-15 papers

bims-toxgon

Biomed News

on Toxoplasma gondii metabolism

Issue of 2023–01–15
nine papers selected by
Lakesh Kumar, BITS Pilani

IMC29 Plays an Important Role in Toxoplasma Endodyogeny and Reveals New Components of the Daughter-Enriched IMC Proteome.
A Microtubule-Associated Protein Is Essential for Malaria Parasite Transmission.
Structure-Function Relationship for a Divergent Atg8 Protein Required for a Nonautophagic Function in Apicomplexan Parasites.
Dynamic-tuning yeast storage carbohydrate improves the production of acetyl-CoA-derived chemicals.
Malaria parasites harness Rho GTPase signaling and host cell membrane ruffling for productive invasion of hepatocytes.
[Evaluation of Ex Vivo Cultivation Potentials of Trypanosoma cruzi, Leishmania tropica ve Toxoplasma gondii Parasites in J774, Vero and HeLa Cell Lines].
The Importance of mTORC1-Autophagy Axis for Skeletal Muscle Diseases.
The effects of L-citrulline adjunctive treatment of Toxoplasma gondii RH strain infection in a mouse model.
Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli.

mBio. 2023 Jan 09. e0304222

IMC29 Plays an Important Role in Toxoplasma Endodyogeny and Reveals New Components of the Daughter-Enriched IMC Proteome.

Peter S Back, Andy S Moon, Rebecca R Pasquarelli, Hannah N Bell, Juan A Torres, Allan L Chen, Jihui Sha, Ajay A Vashisht, James A Wohlschlegel, Peter J Bradley.

  The Toxoplasma inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δimc29 parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle. IMPORTANCE The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.

Keywords:  BioID; Toxoplasma gondii; coiled-coil domains; inner membrane complex; parasitology; replication

DOI:  https://doi.org/10.1128/mbio.03042-22
mBio. 2023 Jan 10. e0331822

A Microtubule-Associated Protein Is Essential for Malaria Parasite Transmission.

Jan Stephan Wichers-Misterek, Annika M Binder, Paolo Mesén-Ramírez, Lilian Patrick Dorner, Soraya Safavi, Gwendolin Fuchs, Tobias L Lenz, Anna Bachmann, Danny Wilson, Friedrich Frischknecht, Tim-Wolf Gilberger.

  Mature gametocytes of Plasmodium falciparum display a banana (falciform) shape conferred by a complex array of subpellicular microtubules (SPMT) associated with the inner membrane complex (IMC). Microtubule-associated proteins (MAPs) define MT populations and modulate interaction with pellicular components. Several MAPs have been identified in Toxoplasma gondii, and homologues can be found in the genomes of Plasmodium species, but the function of these proteins for asexual and sexual development of malaria parasites is still unknown. Here, we identified a novel subpellicular MAP, termed SPM3, that is conserved within the genus Plasmodium, especially within the subgenus Laverania, but absent in other Apicomplexa. Conditional knockdown and targeted gene disruption of Pfspm3 in Plasmodium falciparum cause severe morphological defects during gametocytogenesis, leading to round, nonfalciform gametocytes with an aberrant SPMT pattern. In contrast, Pbspm3 knockout in Plasmodium berghei, a species with round gametocytes, caused no defect in gametocytogenesis, but sporozoites displayed an aberrant motility and a dramatic defect in invasion of salivary glands, leading to a decreased efficiency in transmission. Electron microscopy revealed a dissociation of the SPMT from the IMC in Pbspm3 knockout parasites, suggesting a function of SPM3 in anchoring MTs to the IMC. Overall, our results highlight SPM3 as a pellicular component with essential functions for malaria parasite transmission. IMPORTANCE A key structural feature driving the transition between different life cycle stages of the malaria parasite is the unique three-membrane pellicle, consisting of the parasite plasma membrane (PPM) and a double membrane structure underlying the PPM termed the inner membrane complex (IMC). Additionally, there are numerous linearly arranged intramembranous particles (IMPs) linked to the IMC, which likely link the IMC to the subpellicular microtubule cytoskeleton. Here, we identified, localized, and characterized a novel subpellicular microtubule-associated protein unique to the genus Plasmodium. The knockout of this protein in the human-pathogenic species P. falciparum resulted in malformed gametocytes and aberrant microtubules. We confirmed the microtubule association in the P. berghei rodent malaria homologue and show that its knockout results in a perturbed microtubule architecture, aberrant sporozoite motility, and decreased transmission efficiency.

Keywords:  Plasmodium falciparum; gametocytogenesis; malaria; microtubule

DOI:  https://doi.org/10.1128/mbio.03318-22
mBio. 2023 Jan 10. e0364221

Structure-Function Relationship for a Divergent Atg8 Protein Required for a Nonautophagic Function in Apicomplexan Parasites.

Marta Walczak, Thomas R Meister, Hoa Mai Nguyen, Yili Zhu, Sébastien Besteiro, Ellen Yeh.

  Atg8 family proteins are highly conserved eukaryotic proteins with diverse autophagy and nonautophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well established, little is known about the molecular changes that facilitated acquisition of divergent, nonautophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study nonautophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here, we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of the Atg8 protein family, is required for P. falciparum Atg8 (PfAtg8) apicoplast localization and function, likely via Atg8 lipidation. On the other hand, another region previously implicated in canonical Atg8 interactions, the N-terminal helix, is not required for apicoplast-specific PfAtg8 function. Finally, our investigations at the cellular level demonstrate that the unique apicomplexan-specific loop, previously implicated in interaction with membrane conjugation machinery in recombinant protein-based in vitro assays, is not required for membrane conjugation nor for the apicoplast-specific effector function of Atg8 in both P. falciparum and related Apicomplexa member Toxoplasma gondii. These results suggest that the effector function of apicomplexan Atg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions. IMPORTANCE The most extensively studied role of Atg8 proteins is in autophagy. However, it is clear that they have other nonautophagic functions critical to cell function and disease pathogenesis that are so far understudied compared to their canonical role in autophagy. Mammalian cells contain multiple Atg8 paralogs that have diverse, specialized functions. Gaining molecular insight into their nonautophagic functions is difficult because of redundancy between the homologs and their role in both autophagy and nonautophagic pathways. Malaria parasites such as Plasmodium falciparum are a unique system to study a novel, nonautophagic function of Atg8 separate from its role in autophagy: they have only one Atg8 protein whose only essential function is in the inheritance of the apicoplast, a unique secondary plastid organelle. Insights into the molecular basis of PfAtg8's function in apicoplast biogenesis will have important implications for the evolution of diverse nonautophagic functions of the Atg8 protein family.

Keywords:  Atg8; Plasmodium; Toxoplasma; apicomplexan parasites; apicoplast; malaria; nonautophagic function of Atg8

DOI:  https://doi.org/10.1128/mbio.03642-21
iScience. 2023 Jan 20. 26(1): 105817

Dynamic-tuning yeast storage carbohydrate improves the production of acetyl-CoA-derived chemicals.

Chenxi Qiu, Huilin Tao, Yu Shen, Qingsheng Qi, Jin Hou.

  Acetyl-coenzyme A (Acetyl-CoA) and malonyl-coenzyme A (malonyl-CoA) are important precursors for producing various chemicals, and their availability affects the production of their downstream chemicals. Storage carbohydrates are considered important carbon and energy reservoirs. Herein, we find that regulating the storage carbohydrate synthesis improves metabolic fluxes toward malonyl-CoA. Interestingly, not only directly decreasing storage carbohydrate accumulation improved malonyl-CoA availability but also increasing the storage carbohydrate by UGP1 overexpression enables an even higher production of acetyl-CoA- and malonyl-CoA-derived chemicals. We find that Ugp1p overexpression dynamically regulates the carbon flux to storage carbohydrate synthesis. In early exponential phases, Ugp1 overexpression causes more storage carbohydrate accumulation, while the carbon flux is then redirected toward acetyl-CoA and malonyl-CoA in later phases, thereby contributing to the synthesis of their derived products. Our study demonstrates the importance of storage carbohydrates rearrangement for the availability of acetyl-CoA and malonyl-CoA and therefore will facilitate the synthesis of their derived chemicals.

Keywords:  Biochemistry; Biomolecules; Biotechnology

DOI:  https://doi.org/10.1016/j.isci.2022.105817
Cell Rep. 2022 Dec 30. pii: S2211-1247(22)01828-9. [Epub ahead of print]42(1): 111927

Malaria parasites harness Rho GTPase signaling and host cell membrane ruffling for productive invasion of hepatocytes.

Antonino Schepis, Sudhir Kumar, Stefan H I Kappe.

  Plasmodium sporozoites are the motile forms of the malaria parasites that infect hepatocytes. The initial invasion of hepatocytes is thought to be actively driven by sporozoites, but host cell processes might also play a role. Sporozoite invasion triggers a host plasma membrane invagination that forms a vacuole around the intracellular parasite, which is critical for subsequent intracellular parasite replication. Using fast live confocal microscopy, we observed that the initial interactions between sporozoites and hepatocytes induce plasma membrane ruffles and filopodia extensions. Importantly, we find that these host cell processes facilitate invasion and that Rho GTPase signaling, which regulates membrane ruffling and filopodia extension, is critical for productive infection. Interestingly, sporozoite cell traversal stimulates these processes, suggesting that it increases hepatocyte susceptibility to productive infection. Our study identifies host cell signaling events involved in plasma membrane dynamics as a critical host component of successful malaria parasite infection of hepatocytes.

Keywords:  Apicomplexa; CP: Cell biology; CP: Microbiology; Hepatocyte; Liver Stage; Malaria; Membrane Ruffles; Rho GTPases; Sporozoite

DOI:  https://doi.org/10.1016/j.celrep.2022.111927
Mikrobiyol Bul. 2023 Jan;57(1): 71-82

[Evaluation of Ex Vivo Cultivation Potentials of Trypanosoma cruzi, Leishmania tropica ve Toxoplasma gondii Parasites in J774, Vero and HeLa Cell Lines].

Ahmet Yıldırım, Ahmet Özbilgin, Kor Yereli.

Three obligate intracellular protozoan parasite species, namely Trypanosoma cruzi, Leishmania tropica and Toxoplasma gondii, causative agents of Chagas disease, Leishmaniasis and toxoplasmosis, respectively, which are responsible for significant morbidity and mortality and reside in macrophage cells, affect more than half of the world's population in connection with socio-economic and geographical factors and also causes neglected parasitic diseases of increasing importance. This study aimed to evaluate the ex vivo cultivation potential of T.cruzi, L.tropica and T.gondii parasites in J774, Vero and HeLa cells and to reproduce in a short time and in large amounts without losing their virulence properties. Ex vivo experimental models were created by infecting J774, Vero and HeLa cell lines confluently produced in cell culture flasks with T.cruzi, L.tropica and T.gondii parasites. In ex vivo cultivation, one passage was applied for seven days and three times in a row. Cells removed from the surface after each passage were plated on eight-well chamber slides. Giemsa stained slides were prepared and infection rates were evaluated by light microscopic examination. At the end of the study, it was observed that all three cell lines could be infected with T.cruzi, L.tropica and T.gondii parasites, and infection rates increased in all cell lines after consecutive passages. As a result of ex vivo cultivation, the best cell lines from which T.cruzi and L.tropica strains grew, were J774, Vero and HeLa, and HeLa, J774 and Vero cell lines for T.gondii strain, respectively (p<0.05). Trypanosoma cruzi, L.tropica and T.gondii parasites were successfully grown in J774, Vero and HeLa cell lines by ex vivo culture method in a short time and in large amounts without losing their virulence properties. Cell lines with the best ex vivo cultivation potential for T.cruzi and L.tropica parasites were J774, Vero and HeLa, respectively, while HeLa, J774 and Vero for T.gondii. It is thought that the data obtained in this regard will contribute to many studies on the development of vaccines, drugs and new diagnostic kits.

DOI: https://doi.org/10.5578/mb.20239906
Int J Mol Sci. 2022 Dec 24. pii: 297. [Epub ahead of print]24(1):

The Importance of mTORC1-Autophagy Axis for Skeletal Muscle Diseases.

Xujun Han, Kah Yong Goh, Wen Xing Lee, Sze Mun Choy, Hong-Wen Tang.

  The mechanistic target of rapamycin (mTOR) complex 1, mTORC1, integrates nutrient and growth factor signals with cellular responses and plays critical roles in regulating cell growth, proliferation, and lifespan. mTORC1 signaling has been reported as a central regulator of autophagy by modulating almost all aspects of the autophagic process, including initiation, expansion, and termination. An increasing number of studies suggest that mTORC1 and autophagy are critical for the physiological function of skeletal muscle and are involved in diverse muscle diseases. Here, we review recent insights into the essential roles of mTORC1 and autophagy in skeletal muscles and their implications in human muscle diseases. Multiple inhibitors targeting mTORC1 or autophagy have already been clinically approved, while others are under development. These chemical modulators that target the mTORC1/autophagy pathways represent promising potentials to cure muscle diseases.

Keywords:  autophagy; mTORC1; muscle diseases

DOI:  https://doi.org/10.3390/ijms24010297
Acta Trop. 2023 Jan 10. pii: S0001-706X(23)00017-7. [Epub ahead of print] 106830

The effects of L-citrulline adjunctive treatment of Toxoplasma gondii RH strain infection in a mouse model.

Abeer E Saad, Hager S Zoghroban, Heba B Ghanem, Dina M El-Guindy, Salwa S Younis.

  Toxoplasma gondii is a zoonotic intracellular protozoan parasite and its therapeutic limitations are one of its major problems. L-citrulline is an organic compound that has beneficial effects on many diseases. The purpose of this study was to assess the impact of L-citrulline, alone or in combination with sulfamethoxazole-trimethoprim (SMZ-TMP) on acute toxoplasmosis caused by Toxoplasma gondii RH virulent strain. In our study, 60 Swiss albino mice were divided into two main groups; the control group and the infected treated group, which was subdivided into group IIa: infected treated with L-citrulline, group IIb: infected treated with SMZ-TMP, and group IIc: infected treated with L-citrulline combined with SMZ-TMP. The effects of treatment were assessed by parasitological study, electron microscopic study of tachyzoites, and histopathological study of the liver. Moreover, ELISA measurement of the serum level of Interferon-gamma, Interleukin 10, Nitric oxide, and apoptotic markers was used. It was noticed that L-citrulline combined with SMZ-TMP significantly increased the survival time of infected mice with a significant decrease in the number of tachyzoites compared to the other subgroups. Moreover, it increased the levels of measured cytokines and serum anti-apoptotic proteins Bcl-2 and improved the extent of liver cell damage associated with a decrease in inflammatory infiltration. In conclusion, L-citrulline supplementation was found to be effective against acute toxoplasmosis, especially when combined with SMZ-TMP as it has multifactorial mechanisms; nitric oxide production, anti-inflammatory, anti-apoptotic, and immune stimulator.

Keywords:  Acute toxoplasmosis; Apoptosis; Interferon-gamma; L-citrulline; Nitric oxide; Sulfamethoxazole-trimethoprim

DOI:  https://doi.org/10.1016/j.actatropica.2023.106830
Cell Rep. 2023 Jan 04. pii: S2211-1247(22)01851-4. [Epub ahead of print]42(1): 111950

Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli.

Evgeniya Schastnaya, Peter Francis Doubleday, Luca Maurer, Uwe Sauer.

  Advanced mass spectrometry methods have detected thousands of post-translational phosphorylation and acetylation sites in bacteria, but their functional role and the enzymes catalyzing these modifications remain largely unknown. In addition to enzymatic acetylation, lysine residues can also be chemically acetylated by the metabolite acetyl phosphate. In Escherichia coli, acetylation at over 3,000 sites has been linked to acetyl phosphate, but the functionality of this widespread non-enzymatic acetylation is even less clear than the enzyme-catalyzed one. Here, we investigate the role of acetyl-phosphate-mediated acetylation in E. coli central metabolism. Out of 19 enzymes investigated, only GapA and GpmA are acetylated at high stoichiometry, which inhibits their activity by interfering with substrate binding, effectively reducing glycolysis when flux to or from acetate is high. Extrapolating our results to the whole proteome, maximally 10% of the reported non-enzymatically acetylated proteins are expected to reach a stoichiometry that could inhibit their activity.

Keywords:  CP: Microbiology; mass spectrometry; metabolism; post-translational modifications; post-translational regulation; protein acetylation; systems biology

DOI:  https://doi.org/10.1016/j.celrep.2022.111950