Replacing animal models in fungal pathogen research: An in vitro Titam virulence assay for the human fungal pathogen Cryptococcus neoformans

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Cryptococcus neoformans causes 1 million infections each year and more deaths than TB. While most patients are HIV-positive, 30% have no underlying deficiency. Untreated infections are fatal, requiring combination Amphotericin B and 5-flucytosine induction therapy and long-term Fluconazole. Break-through infections are common, and 67% are transiently Fluconazole resistant. Classically, AMR is caused by heritable mutations in target proteins, however fungal AMR is frequently caused by aneuploidy after drug exposure. The C. neoformans Titan cell is perhaps the most extreme example of this capacity for genome plasticity. Titans form when 5m haploid yeast endoreduplicate DNA, becoming large (>15m), highly polyploid (4C-360C) cells. Titans then produce small, aneuploid daughters that proliferate and return to haploidy. However, unlike drug-induced aneuploidy caused by defects in nuclear or chromosome segregation, our data demonstrate that Titanisation is a regulated morphological transition. Titans therefore represent a host-mediated, inducible path to aneuploidy that underlies fungal drug resistance, a major emerging research area. Despite their importance, no in vitro Titanisation assay exists. Titans are transient and only observed in the lung. Currently, analysis involves infecting large numbers of mice via inhalation of yeast and culling after 7 days for end-point analysis. The number of labs studying Titanisation in vivo more than doubled in the last 5 years and will continue to grow. Manuscripts typically document 100-200 mice, and animal numbers in unpublished experiments are likely to be higher (300-500), further urging development of in vitro models. Therefore, this project will validate an in vitro Titanisation assay developed in my lab that will replace mice and facilitate in vitro modelling of the host environment using standard and emerging tissue culture techniques. Further, this model will improve on in vivo techniques by allowing in vitro observation of Titan cell biology and interaction with host immune cells.