Leveraging an in vitro assay to perform chemical, genomic, and genetic analyses of the molecular mechanisms underpinning the yeast to titan transition in Cryptococcus neoformans

Zafar, Hanna (2023). Leveraging an in vitro assay to perform chemical, genomic, and genetic analyses of the molecular mechanisms underpinning the yeast to titan transition in Cryptococcus neoformans. University of Birmingham. Ph.D.

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Recognition of the threat that fungal pathogens impose on human health is necessary, especially when there is a lack of funding and knowledge behind the species and the diseases they cause. Cryptococcus neoformans infection has increased in occurrence over the past 40 years particularly among the immunocompromised, and yet our understanding of C. neoformans pathogenesis and drug resistance remains limited. Moreover, long-term use of azoles has led to the development of drug resistance via poorly understood mechanisms. During infection, C neoformans undergoes an unusual morphological transition: formation of titan cells during which yeast grow isotropically to over 10-100 μm, forming highly polyploid Titan cells. Previously it was difficult to study the molecular mechanism behind titanisation and its induction due to titan cells only being seen in vivo, making the knowledge behind this quite limited. Now thanks to the development of our in vitro model I can successfully study this phenomenon without the use of mice, and allow for a better understanding of the mechanisms behind this transition. The role of titanisation in drug resistance has not yet been explored.

In this thesis, it was shown through the use of paired naïve and heteroresistant clinical isolates that exposure to fluconazole can impart or remove the capacity to form titan cells. While Titan cell growth itself does not necessarily lead to drug resistance, fluconazole does impact genome plasticity and polyploidy, which in turn contribute to increased resistance in C. neoformans. Alongside this, our study showed how there are many pathways, such as cAMP pathway and production of mitochondrial superoxide, that may be facilitating the way fluconazole affects C. neoformans genome plasticity in turn facilitating this resistance. Reducing titanisation using certain FDA approved drugs appeared to increase fluconazole resistance.

In addition, in silico analyses of the sequences of clinical isolates and mutant libraries were used to explore the mechanisms of titanisation. The results identified an additional pathway, the trehalose biosynthesis pathway, which negatively regulates titanisation through Tps1. To date this pathway has not been known to play a role in this morphological transition.

This work has helped expand our understanding of titan cell formation and its molecular mechanisms whilst also calling for further investigation into the new pathways and the causes of drug resistance.

Type of Work: Thesis (Doctorates > Ph.D.)
Award Type: Doctorates > Ph.D.
Licence: All rights reserved
College/Faculty: Colleges (2008 onwards) > College of Life & Environmental Sciences
School or Department: School of Biosciences
Funders: Other
Other Funders: NC3Rs
Subjects: Q Science > QR Microbiology
URI: http://etheses.bham.ac.uk/id/eprint/12866


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