Titan cells formation inCryptococcus neoformansis finely tuned by environmental conditions and modulated by positive and negative genetic regulators

Abstract

AbstractThe pathogenic fungusCryptococcus neoformansexhibits 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 optimizedin vitroconditions 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 generatedin vitroharbor the main characteristics of titan cells producedin vivoincluding 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 onLMP1andSGF29genes. By screening a gene deletion collection, we also confirmed thatGPR4/5-RIM101, andCAC1genes were required to generate titan cells and that thePKR1,TSP2,USV101genes 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 robustin vitroassay, our results provide new insights into titan cell biogenesis with the identification of multiple important factors/pathways.Author SummaryCryptococcus neoformansis a yeast that is capable of morphological change upon interaction with the host. Particularly, in the lungs of infected mice, a subpopulation of yeast enlarges, producing cells up to 100 µm in cell body diameter – referred to as titan cells. Along with their large size, the titan cells have other unique characteristics such as thickened cell wall, dense capsule, polyploidization, large vacuole with peripheral nucleus and cellular organelles. The generation of a large number of such cells outside the lungs of mice has been described but was not reproducible nor standardized. Here we report standardized, reproducible, robust conditions for generation of titan cells and explored the environmental and genetic factors underlying the genesis of these cells. We showed that titan cells were generated upon stresses such as change in the incubation medium, nutrient deprivation, hypoxia and low pH. Using collections of well characterized reference strains and clinical isolates, we validated with our model that the cAMP/PKA/Rim101 pathway is a major genetic determinant of titan cell formation. This study opens the way for a more comprehensive picture of the ontology of morphological changes inCryptococcus neoformansand its impact on pathobiology of this deadly pathogen.