ORCID
0000-0003-3992-3526
Date of Award
5-9-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Cryptococcus neoformans is an opportunistic fungal pathogen found ubiquitously in the environment. Cryptococcal infection represents a substantial global health burden, particularly in the setting of HIV infection. The most severe form, cryptococcal meningitis, comprises 19% of AIDS-related mortality. An estimated 194,000 cases of cryptococcal meningitis occur each year which lead to approximately 147,000 deaths. This seventy-five percent mortality rate clearly highlights the need for more effective treatment strategies to combat this pathogen. Cryptococcal infection begins when infectious particles are inhaled and deposited into the lungs. In immunocompetent hosts, the immune system can readily kill fungal cells, but in immunocompromised individuals, this initial infection can grow, permitting dissemination to other organs. Cryptococci can cross the blood-brain-barrier and proliferate in the brain, leading to a fungal meningoencephalitis that is fatal without treatment. A critical stage of this infection, and the one most relevant to this study, is the initial encounter between cryptococcal cells and the host immune system. This step determines whether the infection will proceed to its most lethal form. C. neoformans produces a variety of cellular factors that modulate uptake by and survival in host phagocytes. One critical cryptococcal virulence factor is the capsule, a polysaccharide layer surrounding the cell which is enlarged upon entry into the host environment. This process of capsule induction results in a thick protective coating that provides resistance to phagocytosis and killing by macrophages. Capsule is required for cryptococcal virulence, as acapsular strains are avirulent in standard murine infection models. Despite its vital importance for cryptococcal virulence, the processes by which capsule is synthesized, trafficked, and attached are not well understood. To address this gap, I sought to identify novel factors involved in capsule production. I hypothesized that because capsule is a key factor that influences phagocytosis, strains with altered interactions with phagocytes would be enriched for strains with capsule defects. With this rationale, I performed a fluorescence-based quantitative capsule assay to screen a collection of single gene deletion mutants which had been previously implicated in interactions with primary mouse phagocytic cells. From this screen, I identified 131 gene deletion mutants with altered capsule size, comprising 57 hypocapsular and 74 hypercapsular mutants. When combined with the results of the host cell interaction screen, this yielded 45 high priority strains with capsule defects that led to altered phagocytosis by both mouse and human cells. Many of these strains were lacking genes whose products are predicted to be involved in the cryptococcal secretory pathway. I chose one gene, SAC1, for further investigation. SAC1 encodes a phosphoinositol-4-phosphate (PI4P) phosphatase that is involved in turnover of this lipid to facilitate anterograde secretory trafficking. I found that loss of this gene results in hypocapsular cells, which have striking lipid trafficking defects. I also demonstrated that sac1 mutants accumulate PI4P and exhibit reduced secretion of both protein and capsule material. Interestingly, the capsule produced by cells lacking Sac1 has altered composition, which may be caused by the mislocalization of capsule biosynthetic enzymes. I performed further studies to identify whether the role of Sac1 is conserved in different strain backgrounds and to interrogate the biochemical changes to the capsule that occur following loss of Sac1 function. In related studies, I also investigated the early stages of capsule production in a temporal manner to better understand the morphological changes that occur to the cell soon after the capsule induction process begins. I found that the cellular changes that accompany capsule production, including the thickening of the cell wall and capsule, may occur earlier than previously understood. Overall, my work furthers our understanding of the cell biological processes required for both host-fungal interactions and capsule production and provides a foundation for future studies to investigate the intersection of these areas.
Language
English (en)
Chair and Committee
Tamara Doering
Recommended Citation
Gaylord, Elizabeth, "The Cell Biology of Cryptococcal Capsule Production" (2024). Arts & Sciences Electronic Theses and Dissertations. 3024.
https://openscholarship.wustl.edu/art_sci_etds/3024