Date of Award

5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Genetics and Biochemistry

Committee Member

Dr. Lukasz Kozubowski, Committee Chair

Committee Member

Dr. Julia Frugoli

Committee Member

Dr. Meredith Morris

Committee Member

Dr. Michael Sehorn

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that primarily infects the immunocompromised. It is responsible for approximately 180,000 deaths per year in individuals with HIV. Fluconazole (FLC) is a fungistatic drug widely used to treat cryptococcosis and prevent recurrence. C. neoformans exhibits an intrinsic heteroresistance to FLC, which is an emerging barrier to effective treatment especially in resource-limited regions where FLC is often the only therapy available. Previous studies have demonstrated that C. neoformans cells confer resistance to FLC through aneuploid formation. The mechanisms underlying aneuploid formation during FLC treatment are unknown. This dissertation characterizes the effects of the initial exposure to FLC, which is critical to development of aneuploidy and resistance in C. neoformans. It highlights the unique mitotic cycle of C. neoformans, providing evidence that the ploidy increase during FLC treatment is likely due to the progression of nuclear division despite inhibition of cellular growth. It shows FLC inhibits budding and cytokinesis, producing cells with aberrant morphologies that exhibit increased ploidy levels. Furthermore, through cell sorting and microdissection analysis it highlights the role of multibudded cells or “multimeras” in promoting proliferation during FLC therapy. It provides support that FLC produces a widespread increase in ploidy from which aneuploids could then potentially be selected. Additionally, this dissertation further characterizes the phenomenon of heteroresistance through single cell and colony level analyses. Filipin staining displays the unequal depletion of ergosterol between individual cells within a population, highlighting the heterogeneous response within a population to FLC. It further explores the heterogeneity of FLC-treated cells within a population through colony size analysis. Interestingly, factors that influence slower growth promote the proliferation of resistant cells and also the proliferation of cells we refer to as “survivors.” Survivors are characterized by their ability to form small colonies on FLC, while not exhibiting the fitness tradeoff seen in resistant cells. It provides a thorough analysis of the response of C. neoformans to FLC and proposes a model that summarizes the factors that contribute to growth during FLC treatment.

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