Adaptations of Cryptococcus to the host extracellular niche

Cryptococcus is an opportunistic human fungal pathogen with the potential to cause life- threatening infections of the central nervous system. Ubiquitous in the environment, Cryptococcus switches from a saprotrophic lifestyle in the environment to a pathogenic lifestyle in humans by altering key cellular functions required for adaptation and invasion of the host. Once inhaled, the host extracellular niche (particularly the lung mucosa) serves as a modulator for cryptococcal adaptive phenotypes which are critical for survival and proliferation. However, the cellular changes exhibited by Cryptococcus as it responds to the pulmonary environment is poorly understood. The surface of the lung mucosa is heavily loaded with secretions from host lung cells, such as Type I and II epithelial cells as well as alveolar macrophages. Upon inhalation, Cryptococcus comes into close contact with these secretions. The first part of this thesis probed and described the potency of mammalian cells secreted factors (including lung-associated cells) to stimulate phenotypic responses that are associated with Cryptococcus adaptive mechanisms. While C. neoformans responded to these secreted factors with rapid replication, its sister species C. gattii instead demonstrated a high capacity to form enormously enlarged titan-like cells. Within the lungs, Cryptococcus undergoes morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. In the second part of this thesis, a new in vitro titan-induction approach is introduced. Using this in vitro approach, I revealed a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterised strain-specific differences within the clade. In addition, this approach demonstrates for the first time that the cell-cycle-regulated phenotypes: cell size changes, DNA replication and budding, are not always synchronous during titanisation. i Titan cell formation is triggered by host-specific environmental conditions [such as physiological temperature (37°C) and CO2 level (5%) coupled with hypoxia, and nutrient limitation] and modulated by genetic regulators including those associated with cell cycle progression. The last part of this thesis established a strong correlation between progression of the cell cycle phenotypes (cell size, DNA replication and budding) and expression of cell cycle genes and identifies the role of cryptococc