Making Home Sweet and Sturdy:<i>Toxoplasma gondii</i>ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

Tomita, Tadakimi; Sugi, Tatsuki; Yakubu, Rama R.; Tu, Vincent; Ma, Yanfen; Weiss, Louis M.

doi:10.1128/mbio.02048-16

Cited by 60 publications

(81 citation statements)

References 53 publications

(82 reference statements)

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“…mAb recognition was dependent on both Gant2 and Gant3, as well as, as expected, GalE. Since only one pp-αGalNAcT is required to express Tn-antigen, these findings support the model of Tomita et al (2017), consistent with precedent in animals, that Gant2 is a priming pp-αGalNAcT, and Gant3 is a follow-on pp-αGalNAcT that adds to the number of structures. Since N2 is more dependent on Gant2 than on GAnt3, N2 is likely to be assembled on the amplified number of Tn-antigen structures generated by Gant3.…”

Section: Results and Dicussionsupporting

confidence: 82%

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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Section: Results and Dicussionsupporting

confidence: 82%

Enabling tools forToxoplasmaglycobiology

Gas-Pascual¹,

Ichikawa²,

Sheikh³

et al. 2018

Preprint

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“…P values were calculated using Student's t test (*, P Ͻ 0.05; ***, P Ͻ 0.005; NS, not significant). wheat germ agglutinin (WGA) (N-acetylglucosamine or sialic acid), succinylated WGA (s-WGA) (N-acetylglucosamine), soybean agglutinin (␣-helix-or ␤-sheet-linked N-acetyl-D-galactosamine or galactose), and Dolichos biflorus, Vicia villosa, Artocarpus integrifolia, and Helix pomatia agglutinins (N-acetylgalactosamine) (22,24,26). The most widely used cyst wall stain is Dolichos biflorus agglutinin (DBA), which intensely stains the cyst wall of in vitro (31) and in vivo (15,19,23,(50)(51)(52) cysts.…”

Section: Discussionmentioning

confidence: 99%

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Guevara

Fox

Bzik

2020

mSphere

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The glycosylated mucin domain of the Toxoplasma gondii cyst wall glycoprotein CST1 is heavily stained by Dolichos biflorus agglutinin, a lectin that binds to N-acetylgalactosamine. The cyst wall is also heavily stained by the chitin binding lectin succinylated wheat germ agglutinin (s-WGA), which selectively binds to N-acetylglucosamine-decorated structures. Here, we tracked the localization of N-acetylglucosamine-decorated structures that bind to s-WGA in immature and mature in vitro cysts. s-WGA localization was observed at the cyst periphery 6 h after the differentiation of the tachyzoite-stage parasitophorous vacuole. By day 1 and at all later times after differentiation, s-WGA was localized in a continuous staining pattern at the cyst wall. Coinciding with the maturation of the cyst matrix by day 3 of cyst development, s-WGA also localized in a continuous matrix pattern inside the cyst. s-WGA localized in both the outer and inner layer regions of the cyst wall and in a continuous matrix pattern inside mature 7-and 10-day-old cysts. In addition, s-WGA colocalized in the cyst wall with CST1, suggesting that N-acetylglucosamineand N-acetylgalactosamine-decorated molecules colocalized in the cyst wall. In contrast to CST1, GRA4, and GRA6, the relative accumulation of the molecules that bind s-WGA in the cyst wall was not dependent on the expression of GRA2. Our results suggest that GRA2-dependent and GRA2-independent mechanisms regulate the trafficking and accumulation of glycosylated molecules that colocalize in the cyst wall. IMPORTANCE Chronic Toxoplasma gondii infection is maintained in the central nervous system by thick-walled cysts. If host immunity wanes, cysts recrudesce and cause severe and often lethal toxoplasmic encephalitis. Currently, there are no therapies to eliminate cysts, and little biological information is available regarding cyst structure(s). Here, we investigated cyst wall molecules recognized by succinylated wheat germ agglutinin (s-WGA), a lectin that specifically binds to N-acetylglucosamine-decorated structures. N-Acetylglucosamine regulates cell signaling and plays structural roles at the cell surface in many organisms. The cyst wall and cyst matrix were heavily stained by s-WGA in mature cysts and were differentially stained during cyst development. The relative accumulation of molecules that bind to s-WGA in the cyst wall was not dependent on the expression of GRA2. Our findings suggest that glycosylated cyst wall molecules gain access to the cyst wall via GRA2-dependent and GRA2-independent mechanisms and colocalize in the cyst wall.

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“…Tissue cysts are responsible for transmission to new intermediate or definitive hosts [20] and are also important because they are impervious to drugs and can avoid immune-mediated destruction. While drugs likely cannot gain access to the interior of the tissue cyst due to the highly glycosylated cyst wall encapsulating the bradyzoites and because they are intracellular [21], tissue cysts avoid immune responses through mechanisms that are not fully understood.…”

Section: Tissue Cysts and Chronic Infectionsmentioning

confidence: 99%

Brains and Brawn: Toxoplasma Infections of the Central Nervous System and Skeletal Muscle

Wohlfert

Blader

Wilson

2017

Trends in Parasitology

124

123

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Toxoplasma gondii is a widespread parasitic pathogen that infects over a third of the world’s population. Following an acute infection, the parasite can persist within its mammalian host as intraneuronal or intramuscular cysts. Cysts will occasionally reactivate, and depending on the host’s immune status and site of reactivation, encephalitis or myositis can develop. Because these diseases have high levels of morbidity and can be lethal, it is important to understand how Toxoplasma traffics to these tissues, how the immune response controls parasite burden and contributes to tissue damage, and what mechanisms underlie neurological and muscular pathologies that toxoplasmosis patients present with. This review aims to summarize recent important developments addressing these critical topics.

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Making Home Sweet and Sturdy:Toxoplasma gondiippGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity

Cited by 60 publications

References 53 publications

Enabling tools forToxoplasmaglycobiology

Enabling tools forToxoplasmaglycobiology

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

Brains and Brawn: Toxoplasma Infections of the Central Nervous System and Skeletal Muscle

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