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This cyst is cloaked with a glycosylated structure called the cyst wall. Previously, we demonstrated that a mucin-like glycoprotein, CST1, localizes to the cyst wall and confers structural rigidity on brain cysts in a mucin-like domain-dependent manner. The mucin-like domain of CST1 is composed of 20 units of threonine-rich tandem repeats that are<jats:italic>O-<\/jats:italic>GalNAc glycosylated. A family of enzymes termed polypeptide<jats:italic>N<\/jats:italic>-acetylgalactosaminyltransferases (ppGalNAc-Ts) initiates<jats:italic>O<\/jats:italic>-GalNAc glycosylation. To identify which isoforms of ppGalNAc-Ts are responsible for the glycosylation of the CST1 mucin-like domain and to evaluate the function of each ppGalNAc-T in the overall glycosylation of the cyst wall, all five ppGalNAc-T isoforms were deleted individually from the<jats:italic>T.\u00a0gondii<\/jats:italic>genome. The ppGalNAc-T2 and -T3 deletion mutants produced various glycosylation defects on the cyst wall, implying that many cyst wall glycoproteins are glycosylated by T2 and T3. Both T2 and T3 glycosylate the CST1 mucin-like domain, and this glycosylation is necessary for CST1 to confer structural rigidity on the cyst wall. We established that T2 is required for the initial glycosylation of the mucin-like domain and that T3 is responsible for the sequential glycosylation on neighboring acceptor sites, demonstrating hierarchical glycosylation by two distinct initiating and filling-in ppGalNAc-Ts in an intact organism.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold><jats:italic>Toxoplasma gondii<\/jats:italic>is an obligate intracellular parasite that infects a third of the world\u2019s population. It can cause severe congenital disease and devastating encephalitis in immunocompromised individuals. We identified two glycosyltransferases, ppGalNAc-T2 and -T3, which are responsible for glycosylating cyst wall proteins in a hierarchical fashion. This glycosylation confers structural rigidity on the brain cyst. Our studies provide new insights into the mechanisms of<jats:italic>O-<\/jats:italic>GalNAc glycosylation in<jats:italic>T.\u00a0gondii<\/jats:italic>.<\/jats:p>","DOI":"10.1128\/mbio.02048-16","type":"journal-article","created":{"date-parts":[[2017,1,11]],"date-time":"2017-01-11T01:25:30Z","timestamp":1484097930000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":67,"title":["Making Home Sweet and Sturdy:<i>Toxoplasma gondii<\/i>ppGalNAc-Ts Glycosylate in Hierarchical Order and Confer Cyst Wall Rigidity"],"prefix":"10.1128","volume":"8","author":[{"given":"Tadakimi","family":"Tomita","sequence":"first","affiliation":[{"name":"Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tatsuki","family":"Sugi","sequence":"additional","affiliation":[{"name":"Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rama","family":"Yakubu","sequence":"additional","affiliation":[{"name":"Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincent","family":"Tu","sequence":"additional","affiliation":[{"name":"Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanfen","family":"Ma","sequence":"additional","affiliation":[{"name":"Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Louis M.","family":"Weiss","sequence":"additional","affiliation":[{"name":"Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA"},{"name":"Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0020-7519(00)00124-7"},{"key":"e_1_3_2_3_2","first-page":"521","volume-title":"Toxoplasmagondii. 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