Date of Award
12-20-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Cryptococcus neoformans (Cn) is an opportunistic fungal pathogen responsible for over 112,000 deaths each year. This yeast is ubiquitous in the environment, where it is particularly associated with soil, trees, and bird excreta. When a susceptible host inhales infectious particles, Cn can proliferate in the lungs before disseminating to the brain to cause an often-fatal meningoencephalitis. Cryptococcosis primarily afflicts those living with uncontrolled HIV/AIDS, accounting for ~15% of AIDS-related mortality. The antifungals available to treat this devastating disease are few in number, toxic to patients, and largely inaccessible in resource-limited settings. The major virulence factor of Cn is its complex polysaccharide capsule, which the cell builds in response to host-like conditions. Without its capsule, this pathogen is avirulent. However, capsule polysaccharides are not the only glycans that are critical to fungal survival during infection. The cell wall, comprised of various interconnected glycans, protects the cell from stressors in the environment and in the host. When the wall is perturbed, cells often lose the ability to grow at physiological temperature. Cn also makes storage carbohydrates, glycoproteins, and glycolipids, some of which are essential for its virulence. All of these diverse glycans are synthesized by the activities of glycosyltransferases. As their name suggests, these enzymes transfer a mono- or oligosaccharide moiety from a donor to an acceptor substrate, forming a specific glycosidic linkage in the process. While certain telltale motifs can be used to predict that a given gene encodes a glycosyltransferase, sequence cannot be used to predict its specific biochemical activity. Therefore, each glycosyltransferase must be individually characterized to elucidate its enzymatic and cellular roles. In my thesis work, I first screened cryptococcal mutants lacking predicted glycosyltransferase genes for virulence and various in vitro phenotypes (Chapter 2). I then proposed to study three of the resulting hits in-depth. A glycosyltransferase encoded by CKF44_01050 is likely involved in cell wall synthesis; while this protein was ultimately not the focus of my project, some relevant studies are included in Appendices B-D. A mutant lacking CKF44_05293 had a virulence defect in my initial screen. This gene is predicted to encode glycogenin (Glg1), the enzyme that initiates glycogen synthesis. Glycogen is a cytosolic glucose storage molecule that the cell breaks down as needed to liberate glucose for central metabolism. I confirmed that this gene does in fact encode a functional glycogenin by 1) observing reduced glycogen in the mutant compared to WT and 2) observing the rescue of glycogen synthesis when this gene was expressed in a Saccharomyces cerevisiae strain lacking glycogenin activity. I further identified residues within the protein that are important for its function. I also investigated the presence of α-1,4-glucan in the cell wall of Cn, an incidental finding in one previous report. glg1Δ cells lack α-1,4-glucan in their cell wall, suggesting that this material originates from glycogen in the cytosol. The glycosyltransferase encoded by CKF44_02199 shares 35% amino acid identity with cryptococcal glycogenin. Although this mutant was also deficient in glycogen synthesis, this gene does not encode a second glycogenin. Based on my findings, I named this protein Glucan organizing enzyme 1 (Goe1). Electron microscopy revealed that glycogen aggregates, called rosettes, which cluster at the cell periphery in WT cells, were mislocalized in goe1Δ. goe1Δ cells also featured dramatic disorganization in the inner layer of their walls. To understand this anomaly at the glycan level, I studied cell wall fractions of goe1Δ by multiple methods. In the alkali-insoluble fraction, which contains any glycans linked to chitin, goe1Δ cells completely lack β-1,3-glucan. Because β-1,6-glucans link β-1,3-glucan to insoluble chitin, I believe that Goe1 is responsible for the connection between β-1,3- and β-1,6 glucans in the cell wall. As β-1,3-glucan synthesis is a validated target of antifungals, this discovery may have implications for human health. My mutant phenotyping efforts provide a starting point for those interested in studying previously uncharacterized cryptococcal glycosyltransferases. Collectively, my in-depth studies of glycogenin and Goe1 have uncovered mechanisms that explain the relationship between glycogen and the fungal cell wall. While questions remain about its activity, Goe1 is no longer a hypothetical protein but one with multiple intriguing roles in the basic biology of the cryptococcal cell.
Language
English (en)
Chair and Committee
Tamara Doering
Recommended Citation
Loza, Liza, "Discerning the roles of novel glycosyltransferases in the pathogenic yeast Cryptococcus neoformans" (2023). Arts & Sciences Electronic Theses and Dissertations. 3214.
https://openscholarship.wustl.edu/art_sci_etds/3214