{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T18:40:45Z","timestamp":1787942445042,"version":"build-2784847793"},"reference-count":29,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2022,7,5]],"date-time":"2022-07-05T00:00:00Z","timestamp":1656979200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Kosciuszko Foundation. The American Centre of Polish Culture"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Opportunistic pathogens of the anamorphic genus Cryptococcus are unique considering their virulence factors that in the context of pathogenesis allowed them to achieve evolutionary success. Morphological transformation into giant (Titan) cells is one of the factors contributing to cryptococcosis. Recently established in vitro protocols demonstrate that 5 or 10% fetal bovine serum (FBS) combined with 5% CO2, 37 \u00b0C, and sufficiently low cell density, triggers cellular enlargement (Serum protocols). However, the FBS components that promote this morphological transition remain incompletely characterized. In search of minimal conditions necessary for stimulating the formation of Titan cells, we performed a study where we eliminated serum from the protocol (Serum-free protocol) and instead systematically adjusted the amount of glucose, source of nitrogen (ammonium sulfate), and the pH. We found that exposing cells to PBS with adjusted pH to 7.3, and supplemented with 0.05% glucose, 0.025% ammonium sulfate, 0.004% K2HPO4, 0.0035% MgSO4, in the presence of 5% CO2 at 37 \u00b0C triggers Titan-like cell formation to the same degree as the previously established protocol that utilized 10% FBS as the sole nutrient source. Titan-like cells obtained according to this Serum-free protocol were characterized by cell body size over ten microns, a single enlarged vacuole, thick cell wall, extensive polysaccharide capsule, and changes in the level of cell ploidy, all currently known hallmarks of Titan cells found in vivo. Strikingly, we found that in both, Serum and Serum-free protocols, an optimal pH for Titan-like cell development is ~7.3 whereas relatively acidic pH (5.5) prevents this morphological transition and promotes robust proliferation, while alkaline pH (~8.0) has a profound growth inhibitory effect. Our study demonstrates a critical role of pH response to the formation of Titan cells and indicates that conditions that allow restricted proliferation in the presence of 5% CO2 are sufficient for this morphological transition to form enlarged cells in Cryptococcus neoformans and Cryptococcus gattii species complex.<\/jats:p>","DOI":"10.3390\/pathogens11070768","type":"journal-article","created":{"date-parts":[[2022,7,6]],"date-time":"2022-07-06T05:41:06Z","timestamp":1657086066000},"page":"768","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Establishing Minimal Conditions Sufficient for the Development of Titan-like Cells in Cryptococcus neoformans\/gattii Species Complex"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2062-6169","authenticated-orcid":false,"given":"Mariusz","family":"Dyl\u0105g","sequence":"first","affiliation":[{"name":"Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA"},{"name":"Department of Mycology and Genetics, Faculty of Biological Sciences, University of Wroclaw, 51-148 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodney","family":"Col\u00f3n-Reyes","sequence":"additional","affiliation":[{"name":"Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9629-1169","authenticated-orcid":false,"given":"Yaliz","family":"Loperena-\u00c1lvarez","sequence":"additional","affiliation":[{"name":"Department of Science, Pontifical Catholic University of Puerto Rico-Mayag\u00fcez Campus, Mayag\u00fcez PR 00681, Puerto Rico"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lukasz","family":"Kozubowski","sequence":"additional","affiliation":[{"name":"Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,7,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e00357-16","DOI":"10.1128\/mSphere.00357-16","article-title":"The Case for Adopting the \u2018Species Complex\u2019 Nomenclature for the Etiologic Agents of Cryptococcosis","volume":"2","author":"Bennett","year":"2017","journal-title":"mSphere"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e00238-17","DOI":"10.1128\/mSphere.00238-17","article-title":"Importance of Resolving Fungal Nomenclature: The Case of Multiple Pathogenic Species in the Cryptococcus Genus","volume":"2","author":"Hagen","year":"2017","journal-title":"mSphere"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"873","DOI":"10.1016\/S1473-3099(17)30243-8","article-title":"Global Burden of Disease of HIV-Associated Cryptococcal Meningitis: An Updated Analysis","volume":"17","author":"Rajasingham","year":"2017","journal-title":"Lancet. 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