Enabling tools forToxoplasmaglycobiology

Abstract

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.

N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required forToxoplasma gondiireplication and invasion

“…The synthesis of GPI-anchors, N-glycans and Oglycans requires as donor substrate UDP-GlcNAc, or UDP-GalNAc. The latter can be synthesised from UDP-GlcNAc via GalE [7], a UDP-Glc/UDP-Gal epimerase. In the amino sugar biosynthesis pathway, the glucosamine 6-phosphate N-acetyltransferase, GNA1, catalyses the acetylation of GlcN6P (Fig 1B).…”

“…The endomembrane system of T. gondii is rich in glycoconjugates which play fundamental roles in infectivity, survival, and virulence [7]. Several glycan structures have been characterized in T. gondii including N-glycans [8], O-glycans [9][10][11][12][13], Cmannose [9,14], GPI-anchors [15,16], and others [7].…”

“…The endomembrane system of T. gondii is rich in glycoconjugates which play fundamental roles in infectivity, survival, and virulence [7]. Several glycan structures have been characterized in T. gondii including N-glycans [8], O-glycans [9][10][11][12][13], Cmannose [9,14], GPI-anchors [15,16], and others [7]. These glycans serve various critical functions from invasion to O2 sensing and nutrient storage, hence contributing to the overall virulence of the parasite [7].…”

“…Several glycan structures have been characterized in T. gondii including N-glycans [8], O-glycans [9][10][11][12][13], Cmannose [9,14], GPI-anchors [15,16], and others [7]. These glycans serve various critical functions from invasion to O2 sensing and nutrient storage, hence contributing to the overall virulence of the parasite [7]. Additionally, glycans are critical components of the bradyzoite cyst wall and the disruption of their formation impairs the parasite's ability to persist [17,18].…”

“…Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) serves as a donor by GlcNAc-dependent glycosyltransferases for the synthesis of N-glycans, glycosylphosphatidylinositol (GPI) -anchors, glycoinositolphospholipids (GIPLs), and for the glycosylation of other protein acceptors. Given the critical roles of these structures for infectivity of tachyzoites [7,8,16,19], and bradyzoite survival and replication [17,18],…”