scholarly journals Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection

2019 ◽  
Author(s):  
Amy Bergmann ◽  
Katherine Floyd ◽  
Melanie Key ◽  
Carly Dameron ◽  
Kerrick C. Rees ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Phylogenetic Analyses ◽  
Heme Biosynthesis ◽  
Microbial Pathogens ◽  
Biosynthesis Pathway ◽  
Prosthetic Group ◽  
Apicomplexan Parasites ◽  
Pathogenic Microbes ◽  
Living Organisms

Heme, an iron-enclosed organic ring, is essential for virtually all living organisms by serving as a prosthetic group in proteins that function in diverse cellular activities ranging from diatomic gas transport and detection to mitochondrial respiration to detoxification. Cellular heme levels in microbial pathogens can be a composite of endogenous de novo synthesis or exogenous uptake of heme or heme synthesis intermediates1,2. Intracellular pathogenic microbes switch routes for heme supply when heme availability in their replicative environment fluctuates through infections2. Here, we show that the Toxoplasma gondii, an obligate intracellular human pathogen, encodes a functional heme biosynthesis pathway. A chloroplast-derived organelle, termed apicoplast, is involved in the heme production. Genetic and chemical manipulation revealed that de novo heme production is essential for T. gondii intracellular growth and pathogenesis. Surprisingly, the herbicide oxadiazon significantly impaired Toxoplasma growth, consistent with phylogenetic analyses that show T. gondii protoporphyrinogen oxidase is more closely related to plants than mammals. We further improve upon this inhibition by 15-to 25-fold with two oxadiazon derivatives, providing therapeutic proof that Toxoplasma heme biosynthesis is a druggable target. As T. gondii has been used to model other apicomplexan parasites3, our study underscores the utility of targeting heme biosynthesis in other pathogenic apicomplexans.

scholarly journals Toxoplasma gondii acetyl-CoA synthetase is involved in fatty acid elongation (of long fatty acid chains) during tachyzoite life stages

2018 ◽  
Vol 59 (6) ◽  
pp. 994-1004 ◽  
Author(s):  
David Dubois ◽  
Stella Fernandes ◽  
Souad Amiar ◽  
Sheena Dass ◽  
Nicholas J. Katris ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Toxoplasma Gondii ◽  
Isotope Labeling ◽  
De Novo ◽  
Lipid Synthesis ◽  
Minor Effect ◽  
Parasite Line ◽  
Acetyl Coa ◽  
Apicomplexan Parasites ◽  
A Minor

Apicomplexan parasites are pathogens responsible for major human diseases such as toxoplasmosis caused by Toxoplasma gondii and malaria caused by Plasmodium spp. Throughout their intracellular division cycle, the parasites require vast and specific amounts of lipids to divide and survive. This demand for lipids relies on a fine balance between de novo synthesized lipids and scavenged lipids from the host. Acetyl-CoA is a major and central precursor for many metabolic pathways, especially for lipid biosynthesis. T. gondii possesses a single cytosolic acetyl-CoA synthetase (TgACS). Its role in the parasite lipid synthesis is unclear. Here, we generated an inducible TgACS KO parasite line and confirmed the cytosolic localization of the protein. We conducted 13C-stable isotope labeling combined with mass spectrometry-based lipidomic analyses to unravel its putative role in the parasite lipid synthesis pathway. We show that its disruption has a minor effect on the global FA composition due to the metabolic changes induced to compensate for its loss. However, we could demonstrate that TgACS is involved in providing acetyl-CoA for the essential fatty elongation pathway to generate FAs used for membrane biogenesis. This work provides novel metabolic insight to decipher the complex lipid synthesis in T. gondii.

scholarly journals Toxoplasma gondii requires its plant-like heme biosynthesis pathway for infection

2020 ◽  
Vol 16 (5) ◽  
pp. e1008499
Author(s):  
Amy Bergmann ◽  
Katherine Floyd ◽  
Melanie Key ◽  
Carly Dameron ◽  
Kerrick C. Rees ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Heme Biosynthesis ◽  
Biosynthesis Pathway

scholarly journals Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis

2019 ◽  
Vol 295 (6) ◽  
pp. 1539-1550 ◽  
Author(s):  
Edwin T. Tjhin ◽  
Jenni A. Hayward ◽  
Geoffrey I. McFadden ◽  
Giel G. van Dooren
Keyword(s):  
Oxygen Consumption ◽  
Toxoplasma Gondii ◽  
Heme Biosynthesis ◽  
Covalent Attachment ◽  
Biosynthesis Pathway ◽  
Extracellular Acidification ◽  
Mitochondrial Oxygen Consumption ◽  
Atp Levels ◽  
Free Heme

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.

scholarly journals A Carbamoyl Phosphate Synthetase II (CPSII) Deletion Mutant of Toxoplasma gondii Induces Partial Protective Immunity in Mice

2021 ◽  
Vol 11 ◽  
Author(s):  
Xunhui Zhuo ◽  
Kaige Du ◽  
Haojie Ding ◽  
Di Lou ◽  
Bin Zheng ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
De Novo ◽  
Parasite Growth ◽  
Pyrimidine Biosynthesis ◽  
Mrna Levels ◽  
Biosynthesis Pathway ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate

Toxoplasma gondii is an obligate intracellular protozoan parasite. T. gondii primarily infection in pregnant women may result in fetal abortion, and infection in immunosuppressed population may result in toxoplasmosis. Carbamoyl phosphate synthetase II (CPSII) is a key enzyme in the de novo pyrimidine-biosynthesis pathway, and has a crucial role in parasite replication. We generated a mutant with complete deletion of CPSII via clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 in type-1 RH strain of T. gondii. We tested the intracellular proliferation of this mutant and found that it showed significantly reduced replication in vitro, though CPSII deletion did not completely stop the parasite growth. The immune responses induced by the infection of RHΔCPSII tachyzoites in mice were evaluated. During infection in mice, the RHΔCPSII mutant displayed notable defects in replication and virulence, and significantly enhanced the survival of mice compared with survival of RH-infected mice. We tracked parasite propagation from ascitic fluid in mice infected with the RHΔCPSII mutant, and few tachyzoites were observed at early infection. We also observed that the RHΔCPSII mutant induced greater accumulation of neutrophils. The mutant induced a higher level of T-helper type-1 cytokines [interferon (IFN)-γ, interleukin (IL)-12]. The mRNA levels of signal transducer and activator of transcription cellular transcription factor 1 and IFN regulatory factor 8 were significantly higher in the RHΔCPSII mutant-infected group. Together, these data suggest that CPSII is crucial for parasite growth, and that strains lack the de novo pyrimidine biosynthesis pathway and salvage pathway may become a promising live attenuated vaccine to prevent infection with T. gondii.

The source of heme for vascular heme oxygenase II: de novo heme biosynthesis in rat aorta

2004 ◽  
Vol 82 (4) ◽  
pp. 218-224 ◽  
Author(s):  
Kinga Jaronczyk ◽  
Loc Bui ◽  
Jonathan M Soong ◽  
Brian E McLaughlin ◽  
Gerald S Marks ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Heme Oxygenase ◽  
De Novo ◽  
Aminolevulinic Acid ◽  
Soluble Guanylyl Cyclase ◽  
Rat Aorta ◽  
Heme Biosynthesis ◽  
Activity Levels ◽  
Sprague Dawley ◽  
Prosthetic Group

Heme is an essential prosthetic group or substrate for many proteins, including hemoglobin, and hemo enzymes such as nitric oxide synthase, soluble guanylyl cyclase, and heme oxygenase (HO). HO is responsible for the breakdown of heme into equimolar amounts of biliverdin, iron, and carbon monoxide, the latter of which is thought to play a role in the regulation of vascular tone. It is not clear whether the source of heme for cardiovascular functions is derived from uptake from the extracellular milieu or synthesis. In this study, we tested the hypothesis that blood vessels obtain their supply of heme for HO through de novo synthesis. Adult male Sprague–Dawley rat aorta was incubated at 37 °C in Krebs' solution with 1 µM [14C]δ-aminolevulinic acid (ALA). [14C]ALA uptake was linear for about 30 min and reached a plateau at approximately 100 min. The radioactivity was incorporated into porphyrins and heme as determined by esterification of 14C-labelled metabolites and thin-layer chromatography. The first and rate-limiting step of heme biosynthesis is catalyzed by ALA synthase (ALA-S), the activity of which was determined in rat aorta using a radiometric assay, ~250 nmol·(g wet mass)–1·h–1. Inducing HO-1 in rat aorta with S-nitroso-N-acetyl penicil la mine (500 µM) did not increase ALA-S activity as compared with basal activity levels of the enzyme. It appears that there is a sufficient amount of heme available under basal ALA-S activity conditions to meet the increased demand for heme resulting from HO-1 induction. These observations indicate that the complete enzymatic pathway for de novo heme biosynthesis resides in rat aorta and furthermore indicate that de novo heme synthesis is capable of supplying a substantial portion of the heme substrate for HO in the aorta.Key words: heme biosynthesis, vasculature, carbon monoxide, heme oxygenase, δ-aminolevulinic acid synthase.

scholarly journals Heme auxotrophy in abundant aquatic microbial lineages

2021 ◽  
Vol 118 (47) ◽  
pp. e2102750118
Author(s):  
Suhyun Kim ◽  
Ilnam Kang ◽  
Jin-Won Lee ◽  
Che Ok Jeon ◽  
Stephen J. Giovannoni ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Heme Biosynthesis ◽  
Bacterial Group ◽  
Free Living ◽  
Freshwater Habitats ◽  
Domains Of Life ◽  
Living Organisms ◽  
Microbial Groups ◽  
Genome Analyses

Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential for respiring organisms. Several microbial groups are known to lack the de novo heme biosynthetic pathway and therefore require exogenous heme from the environment. These heme auxotroph groups are largely limited to pathogens, symbionts, or microorganisms living in nutrient-replete conditions, whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms. Here, we show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, is auxotrophic for heme, based on the experimental or genomic evidence. We found that two recently cultivated acI isolates require exogenous heme for their growth. One of the cultured acI isolates also exhibited auxotrophy for riboflavin. According to whole-genome analyses, all (n = 20) isolated acI strains lacked essential enzymes necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial groups, including Patescibacteria, Marinisomatota (SAR406), Actinomarinales (OM1), and Marine groups IIb and III of Euryarchaeota. Our findings indicate that heme auxotrophy is a more common phenomenon than previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial diversity.

scholarly journals Heme auxotrophy in abundant aquatic microbial lineages

2021 ◽  
Author(s):  
Suhyun Kim ◽  
Ilnam Kang ◽  
Jin-Won Lee ◽  
Che-Ok Jeon ◽  
Stephen J. Giovannoni ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Heme Biosynthesis ◽  
Free Living ◽  
Group Iii ◽  
Freshwater Habitats ◽  
Domains Of Life ◽  
Living Organisms ◽  
Microbial Groups ◽  
Genome Analyses

Heme, a porphyrin ring complexed with iron, is a metalloprosthetic group of numerous proteins involved in diverse metabolic and respiratory processes across all domains of life, and is thus considered essential for respiring organisms1,2. Several microbial groups are known to lack the de novo heme biosynthetic pathway and therefore require exogenous heme from the environment3. These heme auxotroph groups are largely limited to pathogens4,5, symbionts6,7, or microorganisms living in nutrient-replete conditions8, whereas the complete absence of heme biosynthesis is extremely rare in free-living organisms9. Here, we show that the acI lineage, a predominant and ubiquitous free-living bacterial group in freshwater habitats, is auxotrophic for heme. We found that two recently cultivated acI isolates10 require exogenous heme for their growth. According to whole-genome analyses, all (n=20) isolated acI strains lacked essential enzymes necessary for heme biosynthesis, indicating that heme auxotrophy is a conserved trait in this lineage. Analyses of >24,000 representative genomes for species clusters of the Genome Taxonomy Database (GTDB) revealed that heme auxotrophy is widespread across abundant but not-yet-cultivated microbial groups, including Patescibacteria, Marinisomatota (SAR406), Actinomarinales (OM1), and marine group III Euryarchaeota. Our findings indicate that heme auxotrophy is a more common phenomenon than previously thought, and may lead to use of heme as a growth factor to increase the cultured microbial diversity.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

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