bims-toxgon Biomed News
on Toxoplasma gondii metabolism
Issue of 2023‒06‒11
seven papers selected by
Lakesh Kumar
BITS Pilani

  1. mSphere. 2023 Jun 05. e0015623
      Toxoplasma gondii is an obligate intracellular parasite whose tachyzoite form causes disease via a lytic growth cycle. Its metabolic and cellular pathways are primarily designed to ensure parasite survival within a host cell. But during its lytic cycle, tachyzoites are exposed to the extracellular milieu and prolonged exposure requires activation of stress response pathways that include reprogramming the parasite proteome. Regulation of protein synthesis is therefore important for extracellular survival. We previously reported that in extracellularly stressed parasites, the elongation phase of protein synthesis is regulated by the Toxoplasma oxygen-sensing protein, PHYb. PHYb acts by promoting the activity of elongation factor eEF2, which is a GTPase that catalyzes the transfer of the peptidyl-tRNA from the A site to the P site of the ribosome. In the absence of PHYb, eEF2 is hyper-phosphorylated, which inhibits eEF2 from interacting with the ribosome. eEF2 kinases are atypical calcium-dependent kinases and BLAST analyses revealed the parasite kinase, CDPK3, as the most highly homologous to the Saccharomyces cerevisiae eEF2 kinase, RCK2. In parasites exposed to extracellular stress, loss of CDPK3 leads to decreased eEF2 phosphorylation and enhanced rates of elongation. Furthermore, co-immunoprecipitation studies revealed that CDPK3 and eEF2 interact in stressed parasites. Since CDPK3 and eEF2 normally localize to the plasma membrane and cytosol, respectively, we investigated how the two can interact. We report that under stress conditions, CDPK3 is not N-myristoylated likely leading to its cytoplasmic localization. In summary, we have identified a novel function for CDPK3 as the first protozoan extracellular stress-induced eEF2 kinase.IMPORTANCEAlthough it is an obligate intracellular parasite, Toxoplasma must be able to survive in the extracellular environment. Our previous work indicated that ensuring that elongation continues during protein synthesis is part of this stress response and that this is due to preventing phosphorylation of elongation factor 2. But the identity of the eEF2 kinase has remained unknown in Toxoplasma and other protozoan parasites. Here, we identify CDPK3 as the first protozoan eEF2 kinase and demonstrate that it is part of a stress response initiated when parasites are exposed to extracellular stress. We also demonstrate that CDPK3 engages eEF2 as a result of its relocalization from the plasma membrane to the cytosol.
    Keywords:  Toxoplasma gondii; kinase; oxygen; parasite; protein synthesis
  2. Protein J. 2023 Jun 07.
      Importin α is a nuclear transporter that binds to nuclear localization signals (NLSs), consisting of 7-20 positively charged amino acids found within cargo proteins. In addition to cargo binding, intramolecular interactions also occur within the importin α protein due to binding between the importin β-binding (IBB) domain and the NLS-binding sites, a phenomenon called auto-inhibition. The interactions causing auto-inhibition are driven by a stretch of basic residues, similar to an NLS, in the IBB domain. Consistent with this, importin α proteins that do not have some of these basic residues lack auto-inhibition; a naturally occurring example of such a protein is found in the apicomplexan parasite Plasmodium falciparum. In this report, we show that importin α from another apicomplexan parasite, Toxoplasma gondii, harbors basic residues (KKR) in the IBB domain and exhibits auto-inhibition. This protein has a long, unstructured hinge motif (between the IBB domain and the NLS-binding sites) that does not contribute to auto-inhibition. However, the IBB domain may have a higher propensity to form an α-helical structure, positioning the wild-type KKR motif in an orientation that results in weaker interactions with the NLS-binding site than a KRR mutant. We conclude that the importin α protein from T. gondii shows auto-inhibition, exhibiting a different phenotype from that of P. falciparum importin α. However, our data indicate that T. gondii importin α may have a low strength of auto-inhibition. We hypothesize that low levels of auto-inhibition may confer an advantage to these important human pathogens.
    Keywords:  Auto-inhibition; Hinge; IBB domain; Importin α; Nuclear import pathway; Toxoplasma gondii
  3. bioRxiv. 2023 May 25. pii: 2023.05.22.539837. [Epub ahead of print]
      Toxoplasma gondii is a zoonotic protist pathogen that infects up to 1/3 of the human population. This apicomplexan parasite contains three genome sequences: nuclear (63 Mb); plastid organellar, ptDNA (35 kb); and mitochondrial organellar, mtDNA (5.9 kb of non-repetitive sequence). We find that the nuclear genome contains a significant amount of NUMTs (nuclear DNA of mitochondrial origin) and NUPTs (nuclear DNA of plastid origin) that are continuously acquired and represent a significant source of intraspecific genetic variation. NUOT (nuclear DNA of organellar origin) accretion has generated 1.6% of the extant T. gondii ME49 nuclear genome; the highest fraction ever reported in any organism. NUOTs are primarily found in organisms that retain the non-homologous end-joining repair pathway. Significant movement of organellar DNA was experimentally captured via amplicon sequencing of a CRISPR-induced double-strand break in non-homologous end-joining repair competent, but not ku80 mutant, Toxoplasma parasites. Comparisons with Neospora caninum , a species that diverged from Toxoplasma ∼28 MY ago, revealed that the movement and fixation of 5 NUMTs predates the split of the two genera. This unexpected level of NUMT conservation suggests evolutionary constraint for cellular function. Most NUMT insertions reside within (60%) or nearby genes (23% within 1.5 kb) and reporter assays indicate that some NUMTs have the ability to function as cis-regulatory elements modulating gene expression. Together these findings portray a role for organellar sequence insertion in dynamically shaping the genomic architecture and likely contributing to adaptation and phenotypic changes in this important human pathogen.Significance Statement: This study reveals how DNA located in cellular compartments called organelles can be transferred to the nucleus of the cell and inserted into the nuclear genome of apicomplexan parasite Toxoplasma . Insertions alter the DNA sequence and may lead to significant changes in how genes function. Unexpectedly, we found that the human protist pathogen, Toxoplasma gondii and closely-related species have the largest observed organellar genome fragment content (>11,000 insertion comprising over 1 Mb of DNA) inserted into their nuclear genome sequence despite their compact 65 Mb nuclear genome. Insertions are occurring at a rate that makes them a significant mutational force that deserves further investigation when examining causes of adaptation and virulence of these parasites.
  4. Front Cell Infect Microbiol. 2023 ;13 1175409
      The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a global disease that significantly impacts human health. The clinical manifestations are mainly observed in immunocompromised patients, including ocular damage and neuronal alterations leading to psychiatric disorders. The congenital infection leads to miscarriage or severe alterations in the development of newborns. The conventional treatment is limited to the acute phase of illness, without effects in latent parasites; consequently, a cure is not available yet. Furthermore, considerable toxic effects and long-term therapy contribute to high treatment abandonment rates. The investigation of exclusive parasite pathways would provide new drug targets for more effective therapies, eliminating or reducing the side effects of conventional pharmacological approaches. Protein kinases (PKs) have emerged as promising targets for developing specific inhibitors with high selectivity and efficiency against diseases. Studies in T. gondii have indicated the presence of exclusive PKs without homologs in human cells, which could become important targets for developing new drugs. Knockout of specific kinases linked to energy metabolism have shown to impair the parasite development, reinforcing the essentiality of these enzymes in parasite metabolism. In addition, the specificities found in the PKs that regulate the energy metabolism in this parasite could bring new perspectives for safer and more efficient therapies for treating toxoplasmosis. Therefore, this review provides an overview of the limitations for reaching an efficient treatment and explores the role of PKs in regulating carbon metabolism in Toxoplasma, discussing their potential as targets for more applied and efficient pharmacological approaches.
    Keywords:  chemotherapy; drug targets; kinases; parasite metabolism; toxoplasmosis
  5. Nat Commun. 2023 Jun 05. 14(1): 3265
      Acetyl-CoA utilized by histone acetyltransferases (HAT) for chromatin modification is mainly generated by ATP-citrate lyase (ACL) from glucose sources. How ACL locally establishes acetyl-CoA production for histone acetylation remains unclear. Here we show that ACL subunit A2 (ACLA2) is present in nuclear condensates, is required for nuclear acetyl-CoA accumulation and acetylation of specific histone lysine residues, and interacts with Histone AcetylTransferase1 (HAT1) in rice. The rice HAT1 acetylates histone H4K5 and H4K16 and its activity on H4K5 requires ACLA2. Mutations of rice ACLA2 and HAT1 (HAG704) genes impair cell division in developing endosperm, result in decreases of H4K5 acetylation at largely the same genomic regions, affect the expression of similar sets of genes, and lead to cell cycle S phase stagnation in the endosperm dividing nuclei. These results indicate that the HAT1-ACLA2 module selectively promotes histone lysine acetylation in specific genomic regions and unravel a mechanism of local acetyl-CoA production which couples energy metabolism with cell division.
  6. mSphere. 2023 Jun 05. e0058722
      During invasion, Plasmodium parasites secrete proteins from rhoptry and microneme apical end organelles, which have crucial roles in attaching to and invading target cells. A sporozoite stage-specific gene silencing system revealed that rhoptry neck protein 2 (RON2), RON4, and RON5 are important for sporozoite invasion of mosquito salivary glands. Here, we further investigated the roles of RON4 during sporozoite infection of the liver in vivo. Following intravenous inoculation of RON4-knockdown sporozoites into mice, we demonstrated that sporozoite RON4 has multiple functions during sporozoite traversal of sinusoidal cells and infection of hepatocytes. In vitro infection experiments using a hepatoma cell line revealed that secreted RON4 is involved in sporozoite adhesion to hepatocytes and has an important role in the early steps of hepatocyte infection. In addition, in vitro motility assays indicated that RON4 is required for sporozoite attachment to the substrate and the onset of migration. These findings indicate that RON4 is crucial for sporozoite migration toward and invasion of hepatocytes via attachment ability and motility.IMPORTANCEMalarial parasite transmission to mammals is established when sporozoites are inoculated by mosquitoes and migrate through the bloodstream to infect hepatocytes. Many aspects of the molecular mechanisms underpinning migration and cellular invasion remain largely unelucidated. By applying a sporozoite stage-specific gene silencing system in the rodent malarial parasite, Plasmodium berghei, we demonstrated that rhoptry neck protein 4 (RON4) is crucial for sporozoite infection of the liver in vivo. Combined with in vitro investigations, it was revealed that RON4 functions during a crossing of the sinusoidal cell layer and invading hepatocytes, at an early stage of liver infection, by mediating the sporozoite capacity for adhesion and the onset of motility. Since RON4 is also expressed in Plasmodium merozoites and Toxoplasma tachyzoites, our findings contribute to understanding the conserved invasion mechanisms of Apicomplexa parasites.
    Keywords:  Plasmodium; invasion; malaria; rhoptry; sporozoite
  7. Elife. 2023 Jun 08. pii: e89185. [Epub ahead of print]12
      Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.
    Keywords:  biochemistry; cell biology; chemical biology; none