Dissertations, Theses, and Capstone Projects

Date of Degree

2-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Ruth E. Stark

Committee Members

Anuradha Janakiraman

Peter N. Lipke

Richard S. Magliozzo

Hsin Wang

Subject Categories

Biochemistry | Biology | Biophysics | Chemistry | Microbiology | Structural Biology

Keywords

melanin, solid-state NMR, fungal cell wall, macromolecular assembly, chitin and chitosan, Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a globally distributed opportunistic fungal pathogen and the causative agent of life threatening cryptococcal meningoencephalitis in immunocompromised individuals, resulting in ~180,000 deaths each year worldwide. A primary virulence-associated trait of this organism is the production of melanin. Melanins are a class of diverse pigments produced via the oxidation and polymerization of aromatic ring compounds that have a characteristically complex, heterogenous, and amorphous structure. They are synthesized by representatives of all biological kingdoms and share a multitude of remarkable properties such as the ability to absorb ultraviolet (UV) light and protect against ionizing radiation. Melanin production in fungi contributes to pathogenicity by conferring resistance to environmental assault and interfering with the host immune response. In C. neoformans, melanin pigments are extruded into the cell wall and form strong associations with various key constituents that are essential for its accumulation and in turn, its ability to function as a virulence factor. The most well-established components of this melanization “scaffold” are the polysaccharides chitin and chitosan; despite a body of evidence suggesting that perturbing the biosynthesis of these polysaccharides disrupts melanization and significantly diminishes virulence, elucidating the underlying molecular interactions between chitin/chitosan and the melanin pigment has proven challenging due to the complex and incompletely-defined structures of the resulting macromolecular fungal assemblies. In this work we use solid-state NMR (ssNMR), a technique uniquely suited to study complex and heterogeneous composite materials, to obtain a molecular level characterization of the cellular “scaffold” on which C. neoformans pigments are deposited. We demonstrate that augmenting the relative or total amount of chitin and chitin modulates cell wall architecture and influences both pigment deposition and retention. We additionally illustrate that perturbing chitin/chitosan biosynthesis unfavorably affects cell-wall flexibility and prevents the accumulation of melanin. Moreover, we identify lipids as an additional group of cellular constituents that strongly bind to the melanin pigment and thus could play a role in the melanization process. In sum, our findings support a model in which it is not the formation of melanin pigments that serves as a virulence factor in C. neoformans, but rather the deposition and retention of the pigments within the cell wall, and thus it is the melanized cell wall that is important rather than the melanin itself.

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