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

“…to critically rely on several glycan structures [7][8][9], TgGNA1 (TGGT1_243600) was assigned a positive fitness score (+1.41) in a genome-wide fitness screen [23], indicating its potential dispensability (Fig 1C). This is unexpected considering the crucial role of GNA1 in other organisms [24], including P. falciparum [21].…”

“…Our results reveal that GNA1 depletion leads to a marked drop in GPI anchors, altering the localization of SAG1 and severely impacting host cell invasion and intracellular growth. Furthermore, UDP-GlcNAc, along with other glycosylations, plays a crucial role in the biosynthesis of N-glycans, modifying numerous proteins in the T. gondii secretory pathway [7]. Several studies suggest that N-glycosylation is essential for parasite invasion, motility, and viability [8,23,46].…”

“…Apicomplexan GNA1s exhibit distinct features and conserved motifs, and a recent structural study highlighted the divergent binding sites for GlcN6P and acetyl-CoA in Cryptosporidium parvum GNA1 compared to human GNA1, including important variations in key residues [21]. The key role of the amino sugar pathway for T. gondii viability, and the predicted significance of GPI anchors and GlcNAc-containing glycoconjugates across T. gondii's life cycle [7,22], underscore the potential of GNA1 as a versatile multistage therapeutic target in toxoplasmosis that could be exploited for selective parasite inhibition.…”

“…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],…”

“…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].…”