This Studentship aims to reduce and replace the use of animal models (mouse, rabbit) in studying a novel disease-related phenotype of the human fungal pathogen Cryptococcus neoformans, test previously intractable hypotheses regarding C. neoformans pathogenesis, and ensure continuing impact by embedding 3Rs practices in training the next generation of researchers.
C. neoformans causes up to 1 million infections and 300,000 deaths each year. While most patients are HIV-positive, 30% have no underlying immune deficiency. Untreated infections are uniformly 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, antimicrobial resistance (AMR) is caused by mutations in target proteins; however fungal AMR is frequently also caused by aneuploidy in response to drug or stress exposure. Perhaps the most extreme example of this capacity for genome plasticity is the C. neoformans Titan cell. Titans form when 5um haploid yeast endoreduplicate DNA, becoming large (>15um), highly polyploid (4C-360C) cells. Titans then produce small, aneuploid daughters that proliferate and return to haploidy. However, unlike stress or drug induced changes in ploidy, which result from defects in nuclear or chromosomal segregation, Titanisation is a regulated morphological transition. Titans therefore represent a host-mediated, inducible path to aneuploidy that underlies drug resistance and is associated with poor patient outcome.
Despite their importance, no in vitro Titanisation assay exists. Titans are transient and only observed in the lung and central nervous system (CNS). The current gold standard model requires infecting large numbers of mice (10 per experimental condition) that are then sacrificed for end-point analysis (bronchial lavage). We estimate that 10,000 mice have been used in these studies since 2012. For example, a single study screening 60 fungal mutants required >600 mice. C. neofomans central nervous system dynamics are tested using the rabbit model of cryptococcal meningitis, and the role of Titanisation in CNS infection remains unaddressed. Such experiments are costly and prohibit molecular analyses of Titanisation mechanisms underling AMR and other host-relevant fungal changes. The number of labs studying Titanisation in vivo has more than doubled over 5 years and will continue to increase.
This project addresses these challenges: my lab have developed an in vitro Titanisation assay that is robust, rapid, simple, and inexpensive. Our preliminary data demonstrate that Titan cells can be induced in vitro and that in vitro Titanisation is predictive of in vivo outcome. Robust validation of the assay will allow it to replace animal use for fungal morphology studies and allow Titans to be studied in real time using standard cell biological approaches, improving on existing strategies and ensuring continuing impact.
To achieve this, the student will 1) Fully characterise in vitro Titan inducing conditions, 2) initiate molecular characterisations and develop hypotheses regard mechanisms of Titanisation in vitro, and 3) receive specific training in 3Rs techniques and play a leadership role in disseminating these findings to the broader research community in order to ensure uptake of 3R refinements. Together, these aims will reduce animal usage in the investigation of C. neoformans pathogenesis, facilitate new insights into C. neoformans biology, and underpin researcher engagement with 3Rs best practices in the growing field of medical mycology.
Zafar, H et al. (2019) A titanic drug resistance threat in Cryptococcus neoformans 52:158-164. doi: 10.1016/j.mib.2019.11.001