POFUT2-mediated <i>O</i>-glycosylation of MIC2 is dispensable for <i>Toxoplasma gondii</i> tachyzoites

Khurana, Sachin; Coffey, Michael; John, Alan; Uboldi, Alessandro D.; Huynh, My Hang; Stewart, Rebecca J.; Carruthers, Vern B.; Tonkin, Christopher J.; Goddard‐Borger, Ethan D.; Scott, Nichollas E.

doi:10.1101/391623

Cited by 2 publications

(2 citation statements)

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“…5H), demonstrating by analogy that the core deoxy-Hex residue is ␣Fuc. Two recent preliminary reports confirm this result in Toxoplasma (36,37). The genome harbors a CAZy GT31 gene (TGGT1_239752 or glt2; Table 1) with homology to animal B3GlcT, which catalyzes the addition of the ␤Glc cap at the 3-position of Fuc.…”

Section: Tools For Toxoplasma Glycobiologymentioning

confidence: 61%

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: Tools For Toxoplasma Glycobiologymentioning

confidence: 61%

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Gas-Pascual

Ichikawa

Sheikh

et al. 2019

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…5H), demonstrating by analogy that the core deoxyHex residue is αFuc. Two recent preliminary reports confirm this result in Toxoplasma Khurana et al, 2018). The genome harbors a CAZy GT31 gene (TGGT1_239752 or glt2; Table 1) with homology to animal B3GlcT, which catalyzes addition of the βGlc cap at the 3-position of Fuc.…”

Section: Results and Dicussionmentioning

confidence: 62%

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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POFUT2-mediated O-glycosylation of MIC2 is dispensable for Toxoplasma gondii tachyzoites

Cited by 2 publications

References 54 publications

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

CRISPR/Cas9 and glycomics tools for Toxoplasma glycobiology

Enabling tools forToxoplasmaglycobiology

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