ORCID
https://orcid.org/0000-0002-7813-4880
Date of Award
8-4-2023
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Cryptococcus neoformans is an opportunistic fungal pathogen that has an enormous impact on global health and is the most common cause of meningitis in adults living with HIV in sub-Saharan Africa. With a mortality rate of 10-75%, cryptococcal meningitis results in over 100,000 deaths per year, comprising 18% of all AIDS-related deaths worldwide. These numbers emphasize the need to understand the basic biology and pathogenesis of this fungus. During infection, spores or desiccated yeast cells are inhaled, resulting in primary pulmonary infection. In immunocompetent hosts, these infections are asymptomatic and are either quickly cleared or remain latent. In immunocompromised patients, however, the fungi disseminate from the lungs and cross the blood-brain barrier to enter the central nervous system, resulting in often-fatal meningoencephalitis. The current standard treatment for cryptococcosis includes only three antifungals, two of which, amphotericin B and fluconazole, target ergosterol and its synthesis. Not only is ergosterol of therapeutic importance, it is also a key component in modulating fungal membrane behavior, such as fluidity and permeability, vesicular trafficking, and signal transduction. As the most abundant sterol in fungi, ergosterol and its synthesis has been widely studied using the model yeast Saccharomyces cerevisiae. However, little is known about sterol organization in the context of fungal pathogenesis. I identified a cryptococcal retrograde sterol transporter, Ysp2 (encoded by CKF44_00650). I found that the lack of Ysp2 under host-mimicking conditions leads to abnormal accumulation of ergoste-rol at the plasma membrane, invagination of the plasma membrane, and malformation of the cell wall, which can be functionally rescued by inhibiting ergosterol synthesis with the antifungal drug fluconazole. Intriguingly, abrogation of retrograde sterol transport also led to increased total sterol accumulation. Overall, as a result of perturbed ergosterol distribution and its consequences, ysp2∆ cells cannot survive in physiologically-relevant environments such as host phagocytes and are dramatically attenuated in virulence. Following up on my studies of cells lacking Ysp2, I performed a suppressor screen based on this mutant, with the goal of identifying novel sterol transport or regulation mechanisms. In an initial screen, I isolated and sequenced 18 strains that suppressed the growth defects of ysp2∆ cells on membrane stressors. Among these suppressors, I identified eight genetic alterations that rescue growth of ysp2∆. Many of the affected genes involved in ergosterol synthesis, which confirms that reducing it is a consistent mechanism for rescuing ysp2∆ defects. I expect further iterations of this screen to reveal novel factors that influence ergosterol distribution and may be exploited as targets of antifungal drugs. Collectively, my findings expand our knowledge of cryptococcal biology, underscore the importance of sterol homeostasis in fungal pathogenesis, and open the way to future studies with relevance to human health.
Language
English (en)
Chair and Committee
Tamara Doering
Recommended Citation
Choy, Hau Lam, "Investigating how sterol transport affects survival of the pathogenic fungus Cryptococcus neoformans in host-like conditions" (2023). Arts & Sciences Electronic Theses and Dissertations. 3112.
https://openscholarship.wustl.edu/art_sci_etds/3112