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

Abstract

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.Author SummaryCryptococcus neoformans is 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 in Cryptococcus neoformans and its impact on pathobiology of this deadly pathogen.

The structural unit of melanin in the cell wall of the fungal pathogenCryptococcus neoformans

“…Only one protein, CNAG_05312, had both an Nterminal signal peptide and a GPI-anchor indicative of plasma membrane association. These secreted peptides were previously reported to play critical roles in C. neoformans virulence (61)(62)(63)(64)(65). Taken together, these results revealed that C. neoformans melanin granules are indeed intimately associated with proteins that employ both a conventional and non-conventional secretory pathway for regulation, and which may be involved with structural and/or functional properties of the cryptococcal eumelanin.…”

Intranasal inoculation ofCryptococcus neoformansin mice produces nasal infection with rapid brain dissemination

“…We noted at 24 h post infection there were rare enlarged yeast cells within the nose turbinates and ears (Fig. 4C-G) whose cell body diameter is above 10 μm, and therefore can be consider titan cells (30–32). The finding of titan cells in the nose of mice is concordant with Lima et al ., (33) which found enlarged cells in noses of guinea pigs after some days of infection.…”

Closely related Cryptococcus neoformans strains possess differential virulence both in humans and the mouse inhalation model

“…Virulence did not correlate with the ability to grow in vitro in either nutrient-rich and limited nutrient conditions, at low and high temperatures (30°C and 37°C) (Supplemental Figure 3). Using two previously described methods (13, 14), we found that virulence was also not associated with the ability to form titan cells in vitro (Supplemental Table 1). However, we observed a trend where low virulence strains formed more titan cells than high and intermediate virulence strains when grown in DMEM media supplemented with serum (Table 4).…”

“…To induce the formation of titan cells in vitro , two previously described methods were used (13, 14). For the first method (13), C. neoformans cells from a YPD agar plate were transferred to 10 ml YPD broth medium in a T25cm3 flask (TPP, Switzerland), grown at 30°C for 22 h with shaking (150 rpm), washed with sterile double distilled water and cell number enumerated with a hemocytometer.…”

“…To induce the formation of titan cells in vitro , two previously described methods were used (13, 14). For the first method (13), C. neoformans cells from a YPD agar plate were transferred to 10 ml YPD broth medium in a T25cm3 flask (TPP, Switzerland), grown at 30°C for 22 h with shaking (150 rpm), washed with sterile double distilled water and cell number enumerated with a hemocytometer. Aliquots of 1 × 10 6 cells in 1 ml of minimal media (15 mM glucose, 10 mM MgSO4, 29.4 mM KH2PO4, 13 mM glycine, 3 μM Vit B1, pH5.5) were transferred into 1.5 ml Eppendorf tubes and incubated in a thermomixer at 30°C with shaking (800 rpm) for 48 hours.…”