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

Abstract

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.

What’s New in Cryptococcus gattii: From Bench to Bedside and Beyond

“…Capsule size correlates with virulence, although not case-fatalit [ 61 ]. Notably, C. gattii seems to have a greater propensity to form titan cells [ 62 ]. Titan cells are abnormally sized (>10 µm) yeast cells that have an immunomodulatory role and potential roles in the pathogenesis of cryptococcosis [ 63 ].…”

Opportunistic Pathogens of the Genus Cryptococcus in Louis Pasteur Days and in 200th Anniversary of his Birth

“…It has been shown that this ability to undergo such specific morphological transformation is unique for pathogenic yeasts of the Cryptococcus species complex [30]. It was confirmed then by two independent research centers that, as in the case of members of the C. neoformans species complex, species of the complex C. gattii, were able to form Titan cells, respectively according to commonly known in vitro serum protocol [31] and the newest protocol based on RPMI medium [114]. Moreover, recently it has been shown that strategies for titanization in case of members of C. gattii and C. neoformans species complexes are significantly different.…”

“…While for C. neoformans typical is synchronous occurrence of cells enlargement and polyploidization, in the case of C. gattii we can observe delay in DNA endoreduplication and cell growth to around 30 μm as a haploid cell. Only after ~ 3 days incubation under titanization conditions DNA endoreduplication can be observed, which finally leads to uninucleate polyploid cells [114]. Moreover, this process was equally effective.…”

“…Thus, it has been shown that, contrary to the assumptions made so far, serum is not a necessary and irreplaceable factor inducing the titanization process. This formed the basis for the development of a new serum-independent Titan cell induction protocols [31,114]. In addition to determining the minimum quantitative and qualitative composition of the medium conditioning the formation of giant cells, the key role of the slightly alkaline pH (7.2-7.3) of the liquid medium used in the induction of Titan cells formation has been demonstrated [31].…”

The Cryptococcus gattii species complex: Unique pathogenic yeasts with understudied virulence mechanisms

“…neoformans [ 92 – 96 ]. We and others have recently investigated titan cell formation across the broader Cryptococcus species complex, which has revealed some subtle differences between lineages [ 44 ]. The impact of these differences on the CGSC–host interaction remains largely unknown [ 97 – 99 ].…”