The Cryptococcus neoformans titanide is a pathogenic cell type that arises from typical yeast cells in response to...

Fungal morphogenesis is a major driver of disease outcome. For the opportunistic fungal pathogen Cryptococcus neoformans, extreme morphological heterogeneity within the lung environment drives dissemination, immune evasion, and drug resistance. One such cell type is the small, oval, titanide (2-4 µm). This poorly understood cell type is prevalent in lung histology as well as host-relevant in vitro conditions. In conditions that generate cellular heterogeneity, titanides comprise 4%-10% of cells 24 hours post-induction and by 72 hours are the dominant cell type. Despite their prevalence and the significance of cryptococcal morphogenesis for virulence, key questions pertaining to their origin, fate, and underlying biology remain unstudied. Using TEM, SEM, and fluorescence microscopy we demonstrate that titanides display distinct morphological features; they possess thin cell walls, single birth scars, and capsules, with reduced Pathogen-Associated Molecular Pattern exposure and altered distribution. These features distinguish titanides from previously described C. neoformans 'small cells' such as in vivo seed cells (6 µm, high cell-wall mannan content) and micro cells (round, <1 μm, with thick cell walls) among others. Using microfluidics, we reveal that titanides arise from yeast-like 'typical' mothers, highlighting the morphological plasticity of typical cells in host-relevant conditions. Upon birth, titanides have compact nuclei and do not immediately proliferate, but transfer to fresh media results in rapid swelling and cell division. Consistent with this, titanides are more resistant to some cell wall/membrane stressors and are an effective infectious propagule in a murine model. Together, these findings establish a definition and essential characterisation of the titanide cell type and provide new insight into a key player in C. neoformans cell biology and pathogenesis.