bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2024‒01‒14
fourteen papers selected by
Lakesh Kumar, BITS Pilani

  1. Nat Commun. 2024 Jan 09. 15(1): 379
      In Apicomplexa, rhoptry discharge is essential for invasion and involves an apical vesicle (AV) docking one or two rhoptries to a macromolecular secretory apparatus. Toxoplasma gondii is armed with 10-12 rhoptries and 5-6 microtubule-associated vesicles (MVs) presumably for iterative rhoptry discharge. Here, we have addressed the localization and functional significance of two intraconoidal microtubule (ICMT)-associated proteins instrumental for invasion. Mechanistically, depletion of ICMAP2 leads to a dissociation of the ICMTs, their detachment from the conoid and dispersion of MVs and rhoptries. ICMAP3 exists in two isoforms that contribute to the control of the ICMTs length and the docking of the two rhoptries at the AV, respectively. This study illuminates the central role ICMTs play in scaffolding the discharge of multiple rhoptries. This process is instrumental for virulence in the mouse model of infection and in addition promotes sterile protection against T. gondii via the release of key effectors inducing immunity.
  2. bioRxiv. 2023 Dec 18. pii: 2023.12.18.572150. [Epub ahead of print]
      Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages requires substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct nuclear sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNA seq data show that TgFBXL2 conditional depletion induces the expression of genes necessary for sexual commitment. We suggest that TgFBXL2 is a latent guardian of sexual stage development in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of sexual development.AUTHOR SUMMARY: Toxoplasma gondii is a protozoan parasite that replicates sexually in felids and asexually in nearly all other mammals with each life stage having a specific transcriptional profile. When life stage specific transcription is not properly controlled, the parasite dies and therefore it's important to understand what inhibits expression of sexual stage genes during asexual growth and vice versa. Here we identify a ubiquitin E3 ligase complex that inhibits sexual stage gene expression during asexual growth.
  3. Bio Protoc. 2024 Jan 05. 14(1): e4916
      Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo-like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements. Key features • This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks. • Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH. • Functional maturation occurs over four weeks.
    Keywords:  Bradyzoites; Cell culture; Human myotubes; Tissue cysts; Toxoplasma gondii
  4. PLoS Pathog. 2024 Jan 11. 20(1): e1011710
      Toxoplasma gondii is an obligate intracellular parasite that infects one-third of the world's human population and establishes infection in the brain. Cerebral immune cell infiltration is critical for controlling the parasite, but little is known about the molecular cues guiding immune cells to the brain during infection. Activated astrocytes produce CCL2, a chemokine that mediates inflammatory monocyte recruitment to tissues by binding to the CCR2 receptor. We detected elevated CCL2 production in the brains of C57BL/6J mice by 15 days after T. gondii infection. Utilizing confocal microscopy and intracellular flow cytometry, we identified microglia and brain-infiltrating myeloid cells as the main producers of CCL2 during acute infection, and CCL2 was specifically produced in regions of parasite infection in the brain. In contrast, astrocytes became the dominant CCL2 producer during chronic T. gondii infection. To determine the role of astrocyte-derived CCL2 in mobilizing immune cells to the brain and controlling T. gondii infection, we generated GFAP-Cre x CCL2fl/fl mice, in which astrocytes are deficient in CCL2 production. We observed significantly decreased immune cell recruitment and increased parasite burden in the brain during chronic, but not acute, infection of mice deficient in astrocyte CCL2 production, without an effect on peripheral immune responses. To investigate potential mechanisms explaining the reduced control of T. gondii infection, we analyzed key antimicrobial and immune players in host defense against T. gondii and detected a reduction in iNOS+ myeloid cells, and T. gondii-specific CD4+ T cells in the knockout mice. These data uncover a critical role for astrocyte-derived CCL2 in immune cell recruitment and parasite control in the brain during chronic, but not acute, T. gondii infection.
  5. Nat Commun. 2024 Jan 09. 15(1): 380
      Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.
  6. Cells. 2023 Dec 25. pii: 48. [Epub ahead of print]13(1):
      Sirtuins (SIRT1-7 in mammals) are a family of NAD+-dependent lysine deacetylases and deacylases that regulate diverse biological processes, including metabolism, stress responses, and aging. SIRT7 is the least well-studied member of the sirtuins, but accumulating evidence has shown that SIRT7 plays critical roles in the regulation of glucose and lipid metabolism by modulating many target proteins in white adipose tissue, brown adipose tissue, and liver tissue. This review focuses on the emerging roles of SIRT7 in glucose and lipid metabolism in comparison with SIRT1 and SIRT6. We also discuss the possible implications of SIRT7 inhibition in the treatment of metabolic diseases such as type 2 diabetes and obesity.
    Keywords:  SIRT1; SIRT6; SIRT7; diabetes; obesity; sirtuin
  7. Bioessays. 2024 Jan 12. e2300211
      Efficient management of low energy states is vital for cells to maintain basic functions and metabolism and avoid cell death. While autophagy has long been considered a critical mechanism for ensuring survival during energy depletion, recent research has presented conflicting evidence, challenging the long-standing concept. This recent development suggests that cells prioritize preserving essential cellular components while restraining autophagy induction when cellular energy is limited. This essay explores the conceptual discourse on autophagy regulation during energy stress, navigating through the studies that established the current paradigm and the recent research that has challenged its validity while proposing an alternative model. This exploration highlights the far-reaching implications of the alternative model, which represents a conceptual departure from the established paradigm, offering new perspectives on how cells respond to energy stress.
    Keywords:  AMPK; autophagy; energy stress response; mTOR
  8. bioRxiv. 2023 Dec 23. pii: 2023.12.22.573073. [Epub ahead of print]
      Breast-cancer brain metastasis (BCBM) poses a significant clinical challenge, resulting in an end-stage diagnosis and hindered by limited therapeutic options. The blood-brain barrier (BBB) acts as an anatomical and physiological hurdle for therapeutic compounds, restricting the effective delivery of therapies to the brain. In order to grow and survive in a nutrient-poor environment, tumors in the brain must adapt to their metabolic needs, becoming highly dependent on acetate. These tumors rely on the conversion of acetate to acetyl-CoA by the enzyme Acetyl-CoA synthetase 2 (ACSS2), a key metabolic enzyme involved in regulating fatty acid synthesis and protein acetylation in tumor cells. ACSS2 has emerged as a crucial enzyme required for the growth of tumors in the brain. Here, we utilized a computational pipeline, combining pharmacophore-based shape screen methodology with ADME property predictions to identify novel brain-permeable ACSS2 inhibitors. From a small molecule library, this approach identified 30 potential ACSS2 binders, from which two candidates, AD-5584 and AD-8007, were validated for their binding affinity, predicted metabolic stability, and, notably, their ability to traverse the BBB. We show that treatment of BCBM cells, MDA-MB-231BR, with AD-5584 and AD-8007 leads to a significant reduction in lipid storage, reduction in colony formation, and increase in cell death in vitro . Utilizing an ex vivo orthotopic brain-slice tumor model, we show that treatment with AD-8007 and AD-5584 significantly reduces tumor size and synergizes with radiation in blocking BCBM tumor growth ex vivo. Importantly, we show that following intraperitoneal injections with AD-5584 and AD-8007, we can detect these compounds in the brain, confirming their BBB permeability. Thus, we have identified and validated novel ACSS2 inhibitor candidates for further drug development and optimization as agents for treating patients with breast cancer brain metastasis.
  9. Cancer Res. 2024 Jan 09.
      Mammalian members of the lysyl oxidase (LOX) family of proteins carry a copper-dependent monoamine oxidase domain exclusively within the C-terminal region, which catalyzes ε-amine oxidation of lysine residues of various proteins. However, recent studies have demonstrated that in LOX-like (LOXL) 2-4 the C-terminal canonical catalytic domain and N-terminal scavenger receptor cysteine-rich (SRCR)-repeats domain exhibit lysine deacetylation and deacetylimination catalytic activities. Moreover, the N-terminal SRCR-repeats domain is more catalytically active than the C-terminal oxidase domain. Thus, LOX is the third family of lysine deacetylases in addition to histone-deacetylase and sirtuin families. In this review, we discuss how the LOX family targets different cellular proteins for deacetylation and deacetylimination to control the development and metastasis of cancer.
  10. bioRxiv. 2023 Dec 21. pii: 2023.12.20.572593. [Epub ahead of print]
      Amino acid withdrawal suppresses mTORC1 signaling rapidly, which initiates macroautophagy (herein, autophagy). Prolonged amino acid deprivation, however, leads to partial reactivation of mTORC1 due to the liberation of free amino acids by autophagic proteolysis. We observed impaired reactivation of mTORC1 signaling and increased apoptotic cell death upon prolonged amino acid withdrawal in cells lacking the AMPKα1/α2 catalytic subunits. These findings align well with the role of AMPK in promoting cell survival during energetic stress but oppose the well-documented inhibitory action of AMPK toward mTORC1. AMPK-mediated reactivation of mTORC1 during prolonged amino acid deprivation could be explained, however, if AMPK were required for autophagy. Indeed, a prevailing view posits that activation of AMPK by glucose withdrawal promotes autophagy and mitophagy through multisite phosphorylation of ULK1. When we examined the role of AMPK in autophagy induced by amino acid deprivation, however, we found unexpectedly that autophagy remained unimpaired in cells lacking AMPK α1/α2, as monitored by several autophagic readouts in several cell lines. Moreover, the absence of AMPK increased ULK1 signaling, LC3b lipidation, and lysosomal acidity, and the phosphorylation of ULK1 S555 (an AMPK site proposed to induce autophagy) decreased upon amino acid withdrawal or pharmacological mTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted basal and amino acid withdrawal-induced autophagy. Together our results demonstrate that AMPK suppresses rather than promotes autophagy and supports mTORC1 signaling during prolonged amino acid deprivation, revealing unexpected roles for AMPK in control of these processes.
  11. Microbiol Spectr. 2024 Jan 09. e0313723
      IMPORTANCE: Sirtuins, as a class of histone deacetylases, have been shown to play important roles in various cellular processes in fungi, including asexual development, stress response, and pathogenicity. By investigating the functions of BbHst3 and BbHst4, we have uncovered their critical contributions to important phenotypes in Beauveria bassiana. Deletion of these sirtuin homologs led to reduced conidial yield, increased sensitivity to osmotic and oxidative stresses, impaired DNA damage repair processes, and decreased fungal virulence. Transcriptomic analyses showed differential expression of numerous genes involved in secondary metabolism, detoxification, transporters, and virulence-related factors, potentially uncovering new targets for manipulation and optimization of fungal biocontrol agents. Our study also emphasizes the significance of sirtuins as key regulators in fungal biology and highlights their potential as promising targets for the development of novel antifungal strategies.
    Keywords:  DNA damage response; asexual development; gene transcription; sirtuins; stress tolerance; virulence
  12. Int J Mol Sci. 2023 Dec 29. pii: 453. [Epub ahead of print]25(1):
      AMP-activated protein kinase (AMPK) is the central component of a signalling pathway that senses energy stress and triggers a metabolic switch away from anabolic processes and towards catabolic processes. There has been a prolonged focus in the pharmaceutical industry on the development of AMPK-activating drugs for the treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. However, recent findings suggest that AMPK inhibitors might be efficacious for treating certain cancers, especially lung adenocarcinomas, in which the PRKAA1 gene (encoding the α1 catalytic subunit isoform of AMPK) is often amplified. Here, we study two potent AMPK inhibitors, BAY-3827 and SBI-0206965. Despite not being closely related structurally, the treatment of cells with either drug unexpectedly caused increases in AMPK phosphorylation at the activating site, Thr172, even though the phosphorylation of several downstream targets in different subcellular compartments was completely inhibited. Surprisingly, the two inhibitors appear to promote Thr172 phosphorylation by different mechanisms: BAY-3827 primarily protects against Thr172 dephosphorylation, while SBI-0206965 also promotes phosphorylation by LKB1 at low concentrations, while increasing cellular AMP:ATP ratios at higher concentrations. Due to its greater potency and fewer off-target effects, BAY-3827 is now the inhibitor of choice for cell studies, although its low bioavailability may limit its use in vivo.
    Keywords:  AMP-activated protein kinase; AMPK; BAY-3827; LKB1; SBI-0206965; cancer; compound C; kinase inhibitors
  13. Int J Mol Sci. 2023 Dec 29. pii: 497. [Epub ahead of print]25(1):
      SIRT6 is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, predominantly located in the nucleus, that is involved in the processes of histone modification, DNA repair, cell cycle regulation, and apoptosis. Disturbances in SIRT6 expression levels have been observed in the development and progression of various types of cancer. Therefore, it is important to better understand the role of SIRT6 in biochemical pathways and assign it specific biological functions. This review aims to summarize the role of SIRT6 in carcinogenesis and tumor development. A better understanding of the factors influencing SIRT6 expression and its biological role in carcinogenesis may help to develop novel anti-cancer therapeutic strategies. Moreover, we discuss the anti-cancer effects and mechanism of action of small molecule SIRT6 modulators (both activators and inhibitors) in different types of cancer.
    Keywords:  SIRT6 activators; SIRT6 inhibitors; deacetylation; histone; sirtuins
  14. Cancers (Basel). 2023 Dec 26. pii: 125. [Epub ahead of print]16(1):
      Autophagy-dependent cisplatin resistance poses a challenge in bladder cancer treatment. SIRT1, a protein deacetylase, is involved in autophagy regulation. However, the precise mechanism through which SIRT1 mediates cisplatin resistance in bladder cancer via autophagy remains unclear. In this study, we developed a cisplatin-resistant T24/DDP cell line to investigate this mechanism. The apoptosis rate and cell viability were assessed using flow cytometry and the CCK8 method. The expression levels of the relevant RNA and protein were determined using RT-qPCR and a Western blot analysis, respectively. Immunoprecipitation was utilized to validate the interaction between SIRT1 and Beclin1, as well as to determine the acetylation level of Beclin1. The findings indicated the successful construction of the T24/DDP cell line, which exhibited autophagy-dependent cisplatin resistance. Inhibiting autophagy significantly reduced the drug resistance index of these cells. The T24/DDP cell line showed a high SIRT1 expression level. The overexpression of SIRT1 activated autophagy, thereby further promoting cisplatin resistance in the T24/DDP cell line. Conversely, inhibiting autophagy counteracted the cisplatin-resistance-promoting effects of SIRT1. Silencing SIRT1 led to increased acetylation of Beclin1, the inhibition of autophagy, and a reduction in the cisplatin resistance of the T24/DDP cell line. Introducing a double mutation (lysine 430 and 437 to arginine, 2KR) in Beclin-1 inhibited acetylation and activated autophagy, effectively reversing the decreased cisplatin resistance resulting from SIRT1 silencing. In summary, our study elucidated that SIRT1 promotes cisplatin resistance in human bladder cancer T24 cells through Beclin1-deacetylation-mediated autophagy activation. These findings suggest a potential new strategy for reversing cisplatin resistance in bladder cancer.
    Keywords:  SIRT1; autophagy; bladder cancer; cisplatin resistance; deacetylation