bims-toxgon 2023-03-05 papers

Issue of 2023–03–05
seven papers selected by
Lakesh Kumar, BITS Pilani

Fac Rev. 2023 ;12 1

Advances towards the complete in vitro life cycle of Toxoplasma gondii.

The full life cycle of Toxoplasma gondii cannot be recapitulated in vitro, and access to certain stages, such as mature tissue cysts (bradyzoites) and oocysts (sporozoites), traditionally requires animal experiments. This has greatly hindered the study of the biology of these morphologically and metabolically distinct stages, which are essential for the infection of humans and animals. However, several breakthrough advances have been made in recent years towards obtaining these life stages in vitro, such as the discovery of several molecular factors that induce differentiation and commitment to the sexual cycle, and different culture methods that use, for example, myotubes and intestinal organoids to obtain mature bradyzoites and different sexual stages of the parasite. We review these novel tools and approaches, highlight their limitations and challenges, and discuss what research questions can already be answered with these models. We finally identify future routes for recapitulating the entire sexual cycle in vitro.

Keywords: Toxoplasma gondii; bradyzoites; in vitro model; life cycle; oocysts; organoids; toxoplasmosis

DOI: https://doi.org/10.12703/r/12-1

bioRxiv. 2023 Feb 24. pii: 2023.02.24.529861. [Epub ahead of print]

IFN-λ is protective against lethal oral Toxoplasma gondii infection.

Mateo Murillo-León, Aura M Bastidas-Quintero, Niklas S Endres, Daniel Schnepf, Estefanía Delgado-Betancourt, Annette Ohnemus, Gregory A Taylor, Martin Schwemmle, Peter Staeheli, Tobias Steinfeldt.

Interferons are essential for innate and adaptive immune responses against a wide variety of pathogens. Interferon lambda (IFN-λ) protects mucosal barriers during pathogen exposure. The intestinal epithelium is the first contact site for Toxoplasma gondii ( T. gondii ) with its hosts and the first defense line that limits parasite infection. Knowledge of very early T. gondii infection events in the gut tissue is limited and a possible contribution of IFN-λ has not been investigated so far. Here, we demonstrate with systemic interferon lambda receptor (IFNLR1) and conditional (Villin-Cre) knockout mouse models and bone marrow chimeras of oral T. gondii infection and mouse intestinal organoids a significant impact of IFN-λ signaling in intestinal epithelial cells and neutrophils to T. gondii control in the gastrointestinal tract. Our results expand the repertoire of interferons that contribute to the control of T. gondii and may lead to novel therapeutic approaches against this world-wide zoonotic pathogen.

DOI: https://doi.org/10.1101/2023.02.24.529861

PLoS Pathog. 2023 Feb;19(2): e1011124

Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

Manickam Yogavel, Alexandre Bougdour, Siddhartha Mishra, Nipun Malhotra, Jyoti Chhibber-Goel, Valeria Bellini, Karl Harlos, Benoît Laleu, Mohamed-Ali Hakimi, Amit Sharma.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

DOI: https://doi.org/10.1371/journal.ppat.1011124

Aging (Albany NY). 2023 Feb 26. 15

Isoform-specific effects of neuronal repression of the AMPK catalytic subunit on cognitive function in aged mice.

Xueyan Zhou, Wenzhong Yang, Xin Wang, Tao Ma.

AMP-activated protein kinase (AMPK) functions as a molecular sensor that plays a critical role in maintaining cellular energy homeostasis. Dysregulation of the AMPK signaling has been linked to synaptic failure and cognitive impairments. Our recent study demonstrates abnormally increased AMPK activity in the hippocampus of aged mice. The kinase catalytic subunit of AMPK exists in two isoforms α1 and α2, and their specific roles in aging-related cognitive deficits are unknown. Taking advantage of the unique transgenic mice (AMPKα1/α2 cKO) recently developed by our group, we investigated how isoform-specific suppression of the neuronal AMPKα may contribute to the regulation of cognitive and synaptic function associated with aging. We found that aging-related impairment of long-term object recognition memory was improved with suppression of AMPKα1 but not AMPKα2 isoform. Moreover, aging-related spatial memory deficits were unaltered with suppression of either AMPKα isoform. Biochemical experiments showed that the phosphorylation levels of the eukaryotic initiation factor 2 α subunit (eIF2α) were specifically decreased in the hippocampus of the AMPKα1 cKO mice. We further performed large-scale unbiased proteomics analysis and revealed identities of proteins whose expression is differentially regulated with AMPKα isoform suppression. These novel findings may provide insights into the roles of AMPK signaling pathway in cognitive aging.

Keywords: AMPK; aging; learning and memory; protein synthesis; proteomics

DOI: https://doi.org/10.18632/aging.204554

Case Rep Ophthalmol. 2022 Sep-Dec;13(3):13(3): 751-755

Toxoplasma Neuroretinitis.

Christine Hsu, Sami H Uwaydat, Joseph G Chacko.

Neuroretinitis is an inflammatory condition with rapid unilateral vision loss, optic disc edema, and macular star formation. While neuroretinitis is commonly due to infectious causes such as Bartonella henselae, neuroretinitis due to toxoplasmosis is uncommon. A 29-year-old male presents to our neuro-ophthalmology clinic on December 7, 2021, at the University of Arkansas for Medical Sciences with symptoms of left eye pain and blurred vision. Subsequent workup led to the diagnosis and treatment of toxoplasma neuroretinitis. The fundus exam eventually demonstrated a notable macular star. Treatment was well tolerated, and the patient regained total visual acuity in the affected eye. Toxoplasma neuroretinitis is known for a characteristic appearance of optic disc edema prior to appearance of stellate maculopathy with vitreous inflammation and peripheral chorioretinal scars. Although loss of vision due to toxoplasmosis is rare, it should be included as part of the differential diagnosis with pertinent history.

Keywords: Neuro-ophthalmology; Ocular toxoplasmosis; Optic nerve/neurophthalmology; Toxoplasma gondii; Toxoplasma neuroretinitis

DOI: https://doi.org/10.1159/000526682

Front Bioeng Biotechnol. 2023 ;11 1125544

In vivo implementation of a synthetic metabolic pathway for the carbon-conserving conversion of glycolaldehyde to acetyl-CoA.

Nils Wagner, Frederik Bade, Elly Straube, Kenny Rabe, Cláudio J R Frazão, Thomas Walther.

Ethylene glycol (EG) derived from plastic waste or CO2 can serve as a substrate for microbial production of value-added chemicals. Assimilation of EG proceeds though the characteristic intermediate glycolaldehyde (GA). However, natural metabolic pathways for GA assimilation have low carbon efficiency when producing the metabolic precursor acetyl-CoA. In alternative, the reaction sequence catalyzed by EG dehydrogenase, d-arabinose 5-phosphate aldolase, d-arabinose 5-phosphate isomerase, d-ribulose 5-phosphate 3-epimerase (Rpe), d-xylulose 5-phosphate phosphoketolase, and phosphate acetyltransferase may enable the conversion of EG into acetyl-CoA without carbon loss. We investigated the metabolic requirements for in vivo function of this pathway in Escherichia coli by (over)expressing constituting enzymes in different combinations. Using 13C-tracer experiments, we first examined the conversion of EG to acetate via the synthetic reaction sequence and showed that, in addition to heterologous phosphoketolase, overexpression of all native enzymes except Rpe was required for the pathway to function. Since acetyl-CoA could not be reliably quantified by our LC/MS-method, the distribution of isotopologues in mevalonate, a stable metabolite that is exclusively derived from this intermediate, was used to probe the contribution of the synthetic pathway to biosynthesis of acetyl-CoA. We detected strong incorporation of 13C carbon derived from labeled GA in all intermediates of the synthetic pathway. In presence of unlabeled co-substrate glycerol, 12.4% of the mevalonate (and therefore acetyl-CoA) was derived from GA. The contribution of the synthetic pathway to acetyl-CoA production was further increased to 16.1% by the additional expression of the native phosphate acyltransferase enzyme. Finally, we demonstrated that conversion of EG to mevalonate was feasible albeit at currently extremely small yields.

Keywords: Ara5P-dependent GAA pathway; Escherichia coli; acetyl-CoA; arabinose 5-phosphate; ethylene glycol; glycolaldehyde; synthetic metabolic pathway

DOI: https://doi.org/10.3389/fbioe.2023.1125544

Biochem Pharmacol. 2023 Feb 25. pii: S0006-2952(23)00055-2. [Epub ahead of print] 115464

Glutamine metabolism in breast cancer and possible therapeutic targets.

Shiqi Li, Hui Zeng, Junli Fan, Fubing Wang, Chen Xu, Yirong Li, Jiancheng Tu, Kenneth P Nephew, Xinghua Long.

Cancer is characterized by metabolic reprogramming, which is a hot topic in tumor treatment research. Cancer cells alter metabolic pathways to promote their growth, and the common purpose of these altered metabolic pathways is to adapt the metabolic state to the uncontrolled proliferation of cancer cells. Most cancer cells in a state of nonhypoxia will increase the uptake of glucose and produce lactate, called the Warburg effect. Increased glucose consumption is used as a carbon source to support cell proliferation, including nucleotide, lipid and protein synthesis. In the Warburg effect, pyruvate dehydrogenase activity decreases, thereby disrupting the TCA cycle. In addition to glucose, glutamine is also an important nutrient for the growth and proliferation of cancer cells, an important carbon bank and nitrogen bank for the growth and proliferation of cancer cells, providing ribose, nonessential amino acids, citrate, and glycerin necessary for cancer cell growth and proliferation and compensating for the reduction in oxidative phosphorylation pathways in cancer cells caused by the Warburg effect. In human plasma, glutamine is the most abundant amino acid. Normal cells produce glutamine via glutamine synthase (GLS), but the glutamine synthesized by tumor cells is insufficient to meet their high growth needs, resulting in a "glutamine-dependent phenomenon." Most cancers have an increased glutamine demand, including breast cancer. Metabolic reprogramming not only enables tumor cells to maintain the reduction-oxidation (redox) balance and commit resources to biosynthesis but also establishes heterogeneous metabolic phenotypes of tumor cells that are distinct from those of nontumor cells. Thus, targeting the metabolic differences between tumor and nontumor cells may be a promising and novel anticancer strategy. Glutamine metabolic compartments have emerged as promising candidates, especially in TNBC and drug-resistant breast cancer. In this review, the latest discoveries of breast cancer and glutamine metabolism are discussed, novel treatment methods based on amino acid transporters and glutaminase are discussed, and the relationship between glutamine metabolism and breast cancer metastasis, drug resistance, tumor immunity and ferroptosis are explained, which provides new ideas for the clinical treatment of breast cancer.

Keywords: Amino acid transporters; Breast cancer; Glutaminase; Glutamine metabolism; Immune microenvironment; ferroptosis

DOI: https://doi.org/10.1016/j.bcp.2023.115464