An in vitro method for inducing titan cells reveals novel features of yeast-to-titan switching in the human fungal pathogen Cryptococcus gattii.

Cryptococcosis is a potentially lethal fungal infection of humans caused by organisms within the Cryptococcus neoformans/gattii species complex. Whilst C. neoformans is a relatively common pathogen of immunocompromised individuals, C. gattii is capable of acting as a primary pathogen of immunocompetent individuals. Within the host, both species undergo morphogenesis to form titan cells: exceptionally large cells that are critical for disease establishment. To date, the induction, defining attributes, and underlying mechanism of titanisation have been mainly characterized in C. neoformans. Here, we report the serendipitous discovery of a simple and robust protocol for in vitro induction of titan cells in C. gattii. Using this in vitro approach, we reveal a remarkably high capacity for titanisation within C. gattii, especially in strains associated with the Pacific Northwest Outbreak, and characterise strain-specific differences within the clade. In particular, this approach demonstrates for the first time that cell size changes, DNA amplification, and budding are not always synchronous during titanisation. Interestingly, however, exhibition of these cell cycle phenotypes was correlated with genes associated with cell cycle progression including CDC11, CLN1, BUB2, and MCM6. Finally, our findings reveal exogenous p-Aminobenzoic acid to be a key inducer of titanisation in this organism. Consequently, this approach offers significant opportunities for future exploration of the underlying mechanism of titanisation in this genus. Author summary: Cryptococcus gattii is a fungal pathogen that causes lethal infections in humans and other animals. Upon entry to the lung, some (but not all) cryptococcal cells are induced to become so-called 'titan cells'; huge cells with thickened cell walls, altered capsule and highly duplicated DNA. As a key virulence determinant, Titan cells can manipulate the host immune system (avoiding phagocytosis and triggering a non-protective immune response) and enhance dissemination to the brain. Thus far, the process of forming titan cells has been largely studied in the related species C. neoformans. Here we describe a new in vitro method to induce titan cells in C. gattii. Using this approach, we discovered the novel, asynchronous progression of cell cycle phenotypes during titanisation and showed how titan formation is strongly influenced by population density as well as the external environment; in particular, the presence of the molecule p-aminobenzoic acid. [ABSTRACT FROM AUTHOR]