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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. [ABSTRACT FROM AUTHOR]

Importance: Post-pandemic, it is essential to understand the epidemiology of pediatric acute respiratory tract infections (ARTIs). Our multi-facility study elucidates the outpatient epidemiology of pediatric ARTI using highly multiplexed PCR testing, providing critical insights into the evolving landscape of the etiological agents with a particular focus on the years following the emergence of SARS-CoV-2. Utilizing data from two different multiplex PCR panels, our research provides a comprehensive analysis of respiratory pathogen positivity from 2018 to 2023. Our findings indicate that over half of the annual test results identified at least one pathogen, primarily of viral origin. Intriguingly, despite the surge in testing during the COVID-19 pandemic, pathogen detection rates remain similar to the pre-pandemic era. These data hold significant implications for directing antimicrobial stewardship strategies, curbing unnecessary antibiotic use in pediatric respiratory diseases, and the value of multiplex PCR testing in the outpatient setting among pediatrics., Competing Interests: T.T.T., B.A.N., Z.H., B.H., M.T., and B.W.G. are employees of bioMérieux, Inc.

A special feature of the human fungal pathogen Cryptococcus neoformans is its morphological changes triggered by the interaction with the host. During infection, a specific increase in cell size is observed, particularly in lung tissue, from a typical cell size of 5-7 μm cells to cells larger than 10 μm, dubbed titan cells (TCs). However, the study of this specific cell subpopulation was, until now, only possible via recovery of TCs from lungs of mice during experimental infections where stable and reproducible generation of TCs occurs.The protocol described here generates TCs using in vitro conditions and measures cell size using a rapid, automated method. TC generation in vitro is robust and reproducible, generating yeast cells harboring the same characteristics of TCs generated in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)