Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization

Lobo-Rojas, Ángel E.; González-Marcano, Eglys; Valera-Vera, Edward; Acosta, Héctor; Quiñones, Wilfredo; Burchmore, Richard; Concepción, Juan Luís; Cáceres, Ana J.

doi:10.1016/j.parint.2016.06.008

Cited by 11 publications

(16 citation statements)

References 50 publications

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“…There is a single UDP-sugar pyrophosphorylase (usp) candidate whose score (Ϫ0.9) suggests that it also does not contribute much to fitness. Because this varies from the negative fitness score for Dol-P-Glc synthase, the UDP-GlcNAc pyrophosphorylase may be promiscuous as in Arabidopsis (89), allowing for redundancy. Nevertheless, we have been unsuccessful in disrupting this gene using our double-CRISPR approach.…”

Section: Sugar Precursor Glycogenesmentioning

confidence: 99%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

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Infection with the protozoan parasite Toxoplasma gondii is a major health risk owing to birth defects, its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Unlike most eukaryotes, Toxoplasma propagates in intracellular parasitophorous vacuoles, but like nearly all other eukaryotes, Toxoplasma glycosylates many cellular proteins and lipids and assembles polysaccharides. Toxoplasma glycans resemble those of other eukaryotes, but species-specific variations have prohibited deeper investigations into their roles in parasite biology and virulence. The Toxoplasma genome encodes a suite of likely glycogenes expected to assemble N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their precursors and membrane transporters. To investigate the roles of specific glycans in Toxoplasma, here we coupled genetic and glycomics approaches to map the connections between 67 glycogenes, their enzyme products, the glycans to which they contribute, and cellular functions. We applied a double-CRISPR/Cas9 strategy, in which two guide RNAs promote replacement of a candidate gene with a resistance gene; adapted MS-based glycomics workflows to test for effects on glycan formation; and infected fibroblast monolayers to assess cellular effects. By editing 17 glycogenes, we discovered novel Glc 0-2-Man 6-GlcNAc 2-type N-glycans, a novel HexNAc-GalNAc-mucin-type O-glycan, and Tn-antigen; identified the glycosyltransferases for assembling novel nuclear O-Fuc-type and cell surface Glc-Fuc-type O-glycans; and showed that they are important for in vitro growth. The guide sequences, editing constructs, and mutant strains are freely available to researchers to investigate the roles of glycans in their favorite biological processes. Toxoplasma gondii is a worldwide, obligate intracellular apicomplexan parasite that can infect most nucleated cells of warm-blooded animals (1), with up to 80% of some human populations being seropositive (2). Toxoplasmosis, the disease caused by Toxoplasma, is associated with encephalitis and blindness in individuals whose parasites are reactivated, as can occur in AIDS and other immunosuppressed patients (3). In utero infections can cause mental retardation, blindness, and death (4). Toxoplasma is transmitted by digesting parasites from feline feces (as oocysts) or undercooked meat (as tissue cysts). Once in the host, parasites convert to the tachyzoite form that disseminates to peripheral tissues (e.g. brain, retina, and muscle). The resulting immune response and/or drugs can control tachyzoite replication, but the parasite survives by encysting into slowly growing bradyzoites. Sporadically, burst of cysts allows the parasites to convert to tachyzoites, whose unchecked growth results in cell and tissue damage (5, 6). Currently, no Toxoplasma vaccine exists, anti-toxoplasmosis drugs have severe side effects, and resistance is developing to these drugs (7-11). As individuals remain infected for life, new anti-Toxoplasma drugs a...

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Section: Sugar Precursor Glycogenesmentioning

confidence: 99%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

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“…Alternatively, Gal may be an alternate source of UDP-Glc via the so-called Isselbacher pathway found in plants and other eukaryotic parasites (Lobo-Rojas et al, 2016). The Glc transporter TgGT1 also transports Gal (Blume et al, 2009) and Toxoplasma has predicted galactokinase (GalK; fitness -2.2) and UDP-Glc/UDP-Gal epimerase (GalE; fitness +1.7) enzymes.…”

Section: Cytoplasmic Glycosylation Glycogenesmentioning

confidence: 99%

Enabling tools forToxoplasmaglycobiology

Gas-Pascual¹,

Ichikawa²,

Sheikh³

et al. 2018

Preprint

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Abbreviations: AAL, Aleuria aurantia agglutinin or lectin; Dol-P-sugar, a conjugate of Glc or Man to dolichol phosphate; EIC, extracted ion chromatogram; FBS, fetal bovine serum; GPI, glycophosphatidylinositol; GT, glycosyltransferase; HAc, acetic acid; HdH, a disaccharide consisting of a hexose and a deoxy-hexose residue; HFF, human foreskin fibroblasts; hTERT, HFF immortalized with human telomerase reverse transcriptase; H6N2, an oligosaccharide consisting of 6 hexose (Hex) and 2 Nacetylhexosamine (HexNAc) residues; NHEJ, non-homologous end-joining; nLC-MS/MS, nano-liquid chromatography coupled to multi-dimensional mass spectrometry; mAb, monoclonal antibody; pp-αGalNAcT, UDP-GalNAc-dependent polypeptide α-N-acetyl-D-galactosaminyltransferase; Tn antigen, a structure corresponding to αGalNAc-S/T; TSR, thrombospondin type 1 repeats ABSTRACT Infection by the protozoan parasite Toxoplasma gondii is a major health risk owing to its chronic nature, ability to reactivate to cause blindness and encephalitis, and high prevalence in human populations. Like nearly all eukaryotes, Toxoplasma glycosylates many of its proteins and lipids and assembles polysaccharides. Unlike most eukaryotes, Toxoplasma divides and differentiates in vacuoles within host cells. While their glycans resemble canonical models, they exhibit species-specific variations that have inhibited deeper investigations into their roles in parasite biology and virulence. The genome of Toxoplasma encodes a suite of likely glycogenes expected to assemble a range of N-glycans, O-glycans, a C-glycan, GPI-anchors, and polysaccharides, along with their requisite precursors and membrane transporters. To facilitate genetic approaches to investigate the roles of specific glycans, we mapped probable connections between 59 glycogenes, their enzyme products, and the glycans to which they contribute. We adapted a double-CRISPR/Cas9 strategy and a mass spectrometry-based glycomics workflow to test these relationships, and conducted infection studies in fibroblast monolayers to probe cellular functions. Through the validated disruption of 17 glycogenes, we also discovered novel Glc0-2-Man6-GlcNAc2-type N-glycans, GalNAc2-and Glc-Fuc-type O-glycans, and a nuclear O-Fuc type glycan. We describe the guide sequences, disruption constructs, and mutant strains, which are freely available to practitioners for application in the context of the relational map to investigate the roles of glycans in their favorite biological processes.

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“…The process occurs also within glycosomes of T. cruzi and Leishmania spp., as inferred from the presence of PTS motifs in several of its enzymes ( Sampaio-Guther et al, 2021 ) ( Supplementary Figure S1 and Supplementary Table S1 ). In these latter parasites, galactose can also serve as substrate for the synthesis of nucleotide sugars, because of the presence in their glycosomes of both a galactokinase (phosphorylating the sugar at its C1 position) and an UDP-pyrophosphatase; these enzymes are absent from T. brucei ( Lobo-Rojas et al, 2016 ; Michels et al, 2021 ). Orthologues of several enzymes of nucleotide-sugar biosynthetic enzymes with a PTS can be detected in each trypanosomatid, bodonid and diplonemid analysed, but not in euglenids or Naegleria spp.…”

Section: Introductionmentioning

confidence: 99%

Delineating transitions during the evolution of specialised peroxisomes: Glycosome formation in kinetoplastid and diplonemid protists

Andrade-Alviárez

Bonive-Boscan

Cáceres

et al. 2022

Front. Cell Dev. Biol.

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One peculiarity of protists belonging to classes Kinetoplastea and Diplonemea within the phylum Euglenozoa is compartmentalisation of most glycolytic enzymes within peroxisomes that are hence called glycosomes. This pathway is not sequestered in peroxisomes of the third Euglenozoan class, Euglenida. Previous analysis of well-studied kinetoplastids, the ‘TriTryps’ parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., identified within glycosomes other metabolic processes usually not present in peroxisomes. In addition, trypanosomatid peroxins, i.e. proteins involved in biogenesis of these organelles, are divergent from human and yeast orthologues. In recent years, genomes, transcriptomes and proteomes for a variety of euglenozoans have become available. Here, we track the possible evolution of glycosomes by querying these databases, as well as the genome of Naegleria gruberi, a non-euglenozoan, which belongs to the same protist supergroup Discoba. We searched for orthologues of TriTryps proteins involved in glycosomal metabolism and biogenesis. Predicted cellular location(s) of each metabolic enzyme identified was inferred from presence or absence of peroxisomal-targeting signals. Combined with a survey of relevant literature, we refine extensively our previously postulated hypothesis about glycosome evolution. The data agree glycolysis was compartmentalised in a common ancestor of the kinetoplastids and diplonemids, yet additionally indicates most other processes found in glycosomes of extant trypanosomatids, but not in peroxisomes of other eukaryotes were either sequestered in this ancestor or shortly after separation of the two lineages. In contrast, peroxin divergence is evident in all euglenozoans. Following their gain of pathway complexity, subsequent evolution of peroxisome/glycosome function is complex. We hypothesize compartmentalisation in glycosomes of glycolytic enzymes, their cofactors and subsequently other metabolic enzymes provided selective advantage to kinetoplastids and diplonemids during their evolution in changing marine environments. We contend two specific properties derived from the ancestral peroxisomes were key: existence of nonselective pores for small solutes and the possibility of high turnover by pexophagy. Critically, such pores and pexophagy are characterised in extant trypanosomatids. Increasing amenability of free-living kinetoplastids and recently isolated diplonemids to experimental study means our hypothesis and interpretation of bioinformatic data are suited to experimental interrogation.

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Trypanosoma cruzi contains two galactokinases; molecular and biochemical characterization

Cited by 11 publications

References 50 publications

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Enabling tools forToxoplasmaglycobiology

Delineating transitions during the evolution of specialised peroxisomes: Glycosome formation in kinetoplastid and diplonemid protists

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