Abstract

Glycans play diverse biological roles, ranging from structural and regulatory functions to mediating cellular interactions. For pathogens, they are also often required for virulence and survival in the host. Our interest in glycoconjugates stems from their role in the fungal pathogen Cryptococcus neoformans. This yeast colonizes the lungs and disseminates to the brain of immunocompromised individuals, where it causes a meningoencephalitis that is frequently lethal, killing almost 200,000 people each year. The major virulence factor of this yeast is a polysaccharide capsule that enables it to manipulate the host immune response and resist host antimicrobial defenses.Synthesis of the capsular glycans and other critical glycoconjugates occurs primarily in the secretory compartment, although almost all activated precursors are made in cytosol. This topological problem is resolved by nucleotide sugar transporters (NSTs), which are thus required for such glycosylation. The identity and regulation of the complete set of cryptococcal NSTs, however, remain elusive. This major gap in our knowledge severely limits our understanding of and ability to manipulate critical biosynthetic processes in this important pathogen.Here, we identified three novel NSTs and determined their kinetic profiles and roles in cryptococcal biology. Uut1 is a high-affinity ER-localized UDP-glucuronic acid (UDP-GlcA) transporter, and Uxt1 and Uxt2 are dual UDP-xylose (UDP-Xyl) transporters found in the Golgi apparatus and endoplasmic reticulum, respectively. Mutants lacking these proteins exhibited compositional changes in their glycoconjugates, including the capsule, increased sensitivity to stress, and altered interactions with phagocytes. UDP-GlcA and UDP-Xyl transport activities were also required for full virulence. Interestingly, UDP-Xyl transport was not required for persistence within the host, as the double uxt1Δ uxt2Δ mutant established a chronic infection of the lung and induced delayed formation of tertiary lymphoid tissue. Collectively, this work advanced our understanding of the localization and sequence of glycan biosynthetic events, and their relationship to virulence. It also set the stage for further studies of fundamental glycobiology, cryptococcal biology and pathogenesis, and potential antifungal agents.

Committee Chair

Tamara L. Doering

Committee Members

Stephen M. Beverley, Mario Feldman, Stuart A. Kornfeld, Amanda L. Lewis,

Comments

Permanent URL: https://doi.org/10.7936/8vj5-b221

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

Spring 5-15-2019

Language

English (en)

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Biology Commons

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