Characterization of the WEE1 Homologues and the Investigation of Factors Promoting Cellular Enlargement in Cryptococcus Neoformans
Date of Award
Doctor of Philosophy (PhD)
Biochemistry and Molecular Biology
Cryptococcus neoformans is an opportunistic fungal pathogen, infecting mainly immunocompromised individuals. As the main cause of cryptococcosis, it is responsible for over 180,000 deaths every year. As an environmental yeast, it has unique adaptations that allow it to proliferate in the human host. Among these adaptations its capacity to transition to an extreme phenotype known as Titan cells is of special interest to researchers. With sizes above 10 um and able to reach 70 um or more in cell size. This size is accompanied with a large vacuole, larger polysaccharide capsule, and an increased resistance to fluconazole (FLC). FLC is a fungistatic drug that is widely used to treat cryptococcosis, so any mechanism that confers resistance to fluconazole is of the utmost importance to understand. Protocols to develop titanization in vitro have been developed, allowing the study of Titan cells in a laboratory setting, but questions remain.
This dissertation utilizes two in vitro titanization protocols to answer if only C. neoformans can titanize. Multiple members of the Cryptococcus spp. were subjected to titanization protocols and analyzed to confirm if they had all characteristics described for Titan cells. This dissertation shows that titanization is unique to the members of the C. neoformans and its sister species C. gattii. Other Cryptococcus spp. could generate Titan- like cells but did not exhibit all the defined characteristics. This dissertation also highlights minimum conditions that contribute to titanization in vitro. While previous titanization protocol relied on serum, here we present a serum-free protocol that induces titanization in vitro and allowed us to analyze these Titan cells. In particular pH was found to be of importance for titanization. An optimal pH for titanization was found to be ~7.3, while slightly acidic pH (5.5) prevents titanization but is conducive to proliferation of yeast cells
and a relatively alkaline (8.0) inhibits proliferation. This dissertation establishes that pH response is critical for Titan cell formation, and that conditions that inhibit proliferation are enough for titanization.
Additionally, this dissertation characterizes two cell cycle control kinases, SWE1 and SWE102. These genes are homologs of the cell cycle regulator SWE1 in Saccharomyces cerevisiae. The cell cycle in budding yeast, S. cerevisiae, is regulated by the morphogenesis checkpoint kinase Swe1, which inhibits cyclin dependent kinase (CDK), by phosphorylation, prior to mitosis and during stress. SWE1 and SWE102 were knocked out individually, generating single deletion mutants. These mutants demonstrated that Swe1 and Swe102 can phosphorylate CDK1 in C. neoformans and can recover the phenotype in a S. cerevisiae swe1∆ strain phosphorylating CDK1 in it as well. The single deletion mutants also show increased sensitivity to antifungals, heat stress response, and surprisingly to DNA damage by genotoxic chemicals. Elimination of SWE1 and SWE102 appears to be synthetic lethal. To circumvent this, a copper regulated promoter (CTR4) was introduced as the promoter for Swe1, while Swe102 was knocked out in the same C. neoformans strains. Treating this CTR4 promoter strain and the single deletion mutants with DNA damaging agents resulted in the treated cells becoming resistant to FLC and showing aneuploidy and increase in ploidy levels, both of which have been associated with FLC resistance. The single deletions were able to titanize, with the swe1∆ demonstrating an irregular-looking capsule and swe102∆ shows an elongated cell body phenotype and capsule formation. We provide an analysis of the myriad roles Swe1 and Swe102 have in cell cycle control, stress response, and morphological transitions demonstrating the importance of studying the cell cycle to better understand the pathogenesis of C. neoformans.
Colón Reyes, Rodney J., "Characterization of the WEE1 Homologues and the Investigation of Factors Promoting Cellular Enlargement in Cryptococcus Neoformans" (2022). All Dissertations. 3140.
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