Abstract

Biofilms are communities of bacteria encased in a biopolymer-rich extracellular matrix. Within biofilms, bacteria are protected from antibiotics, enzymatic digestion, and host immune responses, resulting in difficult-to-treat infections. Pseudomonas aeruginosa is an opportunistic pathogen and can cause chronic infections, including lung infections in people with cystic fibrosis (CF), by forming biofilms. Currently, P. aeruginosa biofilm matrices are known to include extracellular DNA (eDNA), proteins, and the exopolysaccharides alginate, Pel, and Psl. Given that P. aeruginosa is associated with persistent and hard-to-treat infections, understanding the composition of its biofilm matrix is crucial. We focused on exopolysaccharides because they are thought to be key structural components and, therefore, generally assumed to be major compositional contributors to the biofilm matrix. However, measuring exopolysaccharide abundance is a technical challenge, and, as such, there is limited robust data to support this notion. Thus, we quantified the levels of exopolysaccharides in biofilms formed by different P. aeruginosa strains. We used a multidisciplinary approach that included tools from microbiology and biophysical chemistry to detect and quantify exopolysaccharides in various P. aeruginosa biofilms. First, we used microbiology tools such as indicator dyes, immunoblots, and scanning electron microscopy (SEM) to visualize and detect exopolysaccharide production. We then used solid-state NMR- based analyses to quantify the percentage of glycans indicative of exopolysaccharides. Applying our multidisciplinary approach, we investigated the composition of sputum from people with CF. Using solid-state NMR and biochemical analyses, we determined the levels of major biomolecules in sputum and identified the glycosylated proteins present. The next part of this thesis applied high-field solid-state NMR to investigate the structure of P. aeruginosa biofilm exopolysaccharides. Although the structures of exopolysaccharides are known, insoluble forms or modified variants may be overlooked when using methods limited by solubility and molecular weight. Thus, using high field (1.1 GHz) solid-state NMR, we analyzed alginate, which is one of the exopolysaccharides produced by P. aeruginosa. Overall, we used an interdisciplinary approach to study the composition of biological samples, including exopolysaccharides and sputum samples from people with CF. Specifically, we determined the abundance of exopolysaccharides in P. aeruginosa and detected and quantified components of sputum samples from people with CF. In addition, we also analyzed the structure of alginate, providing information on the structure and modifications. Altogether, these studies provide compositional information on biofilms and the host that can inform anti-biofilm therapeutics to treat chronic infections caused by P. aeruginosa.

Committee Chair

Courtney Reichhardt

Committee Members

Courtney Reichhardt; Erin Gloag; Meredith Jackrel; Tamara Doering; Tim Wencewicz

Degree

Doctor of Philosophy (PhD)

Author's Department

Chemistry

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

4-21-2026

Language

English (en)

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Available for download on Thursday, April 20, 2028

Included in

Chemistry Commons

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