The pathogenic fungus Cryptococcus neoformans exhibits morphological changes in cell size during lung infection, producing both typical size 5 to 7 μm cells and large titan cells (> 10 μm and up to 100 μm). We found and optimized in vitro conditions that produce titan cells in order to identify the ancestry of titan cells, the environmental determinants, and the key gene regulators of titan cell formation. Titan cells generated in vitro harbor the main characteristics of titan cells produced in vivo including their large cell size (>10 μm), polyploidy with a single nucleus, large vacuole, dense capsule, and thick cell wall. Here we show titan cells derived from the enlargement of progenitor cells in the population independent of yeast growth rate. Change in the incubation medium, hypoxia, nutrient starvation and low pH were the main factors that trigger titan cell formation, while quorum sensing factors like the initial inoculum concentration, pantothenic acid, and the quorum sensing peptide Qsp1p also impacted titan cell formation. Inhibition of ergosterol, protein and nucleic acid biosynthesis altered titan cell formation, as did serum, phospholipids and anti-capsular antibodies in our settings. We explored genetic factors important for titan cell formation using three approaches. Using H99-derivative strains with natural genetic differences, we showed that titan cell formation was dependent on LMP1 and SGF29 genes. By screening a gene deletion collection, we also confirmed that GPR4/5-RIM101, and CAC1 genes were required to generate titan cells and that the PKR1, TSP2, USV101 genes negatively regulated titan cell formation. Furthermore, analysis of spontaneous Pkr1 loss-of-function clinical isolates confirmed the important role of the Pkr1 protein as a negative regulator of titan cell formation. Through development of a standardized and robust in vitro assay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.
Bibliographical noteFunding Information:
BH’s salary was funded by Assistance Publique-Hôpitaux de Paris and Institut Pasteur (Poste d’ accueil APHP/CNRS/Institut Pasteur). http://recherche.aphp.fr/candidatures-internes/ KN grant funding National Institutes of Health, National Institute of Allergy and Infectious Diseases (NIAID) grant R01AI080275. https://www.niaid.nih.gov AC is supported in part by 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733. https://www.niaid.nih.gov CAD and CAC were supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services grant number U19AI110818. https://www.niaid.nih.gov The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We warmly thank Pr James Kronstad and Melissa Caza for sending us their collection of deletion and complemented mutants of the PKR1 gene, as well as their PGal:PKR1 and PGal:PKA conditional mutants. We thank Dr Tihana Bicanic, Shichina Kannmbath, Charles Giamberardino and Jennifer Tenor who kindly provided specific clinical isolates. The authors want to thank Marie Desnos-Ollivier for her help with Sanger sequencing, JL Tinevez for technical assistance in Biostation experiments, Stéphane Dallongeville for help with Icy Software, Stevenn Volant for biostatistics, Pierre-Henri Commere for FACS analysis, Frederique Moyrand for performing PCR and plasmid preparations. We thank Quigly Dragotakes for helping us determining cell and capsule sizes in specific experiments. The authors acknowledge Hiten Madhani and members of his laboratory for the gene deletion collection that has been made available ahead of publication to the scientific community.