Titan cells formation in Cryptococcus neoformans is finely tuned by environmental conditions and modulated by positive and negative genetic regulators
Year: 2018
Type: article
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 Summary Cryptococcus 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.
Source: PLoS Pathogens
Institutions: Centre National de la Recherche Scientifique, Institut Pasteur, Sorbonne Paris Cité, Assistance Publique – Hôpitaux de Paris, Hôpital Saint-Louis, Génomique évolutive, modélisation et santé, Université Paris Diderot, University of Minnesota, Johns Hopkins University, Broad Institute, Duke Medical Center, The University of Queensland
Language: English
Subfield: Epidemiology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $3,043
Awards: G10969003, G2436179761, Synergy of Host Defense Mechanisms in the Lung, G2773611754, G2898696312, Advancing Genomic Technologies to Combat Infectious Disease: Mapping Dynamics within Single Cells, Individual Hosts, and Global Populations, G4243981961, G4500500705, G4559384, G4980442498, G5625980634, Data commons for 'omic, microscopy and analysis core facilities, Protective antibodies for Cryptococcal Infections, Biology of Fungal Melanin, Evolution of Cryptococcus neoformans Strains from Patients with HIV/AIDS, G9000059902, G9000067794, G9000096691, G9000156212, G9000194839, G9000292769, Impact of Cryptococcus titan cells on pathogenesis, Molecular Characterization of C. neoformans Antibodies
OpenAlex ID: W2804701509