Date of Award

Spring 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Yan, Jing

Abstract

Bacterial biofilms are surface-attached communities of bacterial cells enclosed in an extracellular matrix formed on environmental surfaces and host tissues. Biofilm formation represents a common strategy in facilitating host colonization and infection by human pathogens, and the polymeric matrix provides a mechanism for drug and host immune resistance by acting as a barrier around biofilm-dwelling cells. Bacteria often express multiple adhesive proteins (adhesins), but it is often unclear whether adhesins have specialized or redundant roles. Here, we combine mutagenesis, biochemistry, single-cell imaging, and bioinformatics to show how Vibrio cholerae, the causal agent of pandemic cholera, uses two biofilm-specific adhesins Bap1 and RbmC with overlapping but distinct functions to achieve robust adhesion to diverse surfaces. Chapter 2 focuses how Bap1 and RbmC share a conserved sugar binding domain for anchoring to Vibrio polysaccharide, and Chapter 3 examines their glycan-targeting and nonspecific surface-binding domains, revealing their roles in host colonization and environmental adhesion. Collectively, we found Bap1 and RbmC function as a “double-sided tape”: they share a β-propeller domain that binds to the biofilm matrix exopolysaccharide, but have distinct environment-facing domains. Bap1 adheres to lipids and abiotic surfaces, while RbmC mainly mediates binding to host surfaces. Furthermore, both adhesins contribute to adhesion in an enteroid monolayer colonization model. We expect that similar modular domains may be utilized by other pathogens, and this line of research can potentially lead to new biofilm-removal strategies and biofilm-inspired adhesives. In studying V. cholerae biofilm adhesins, we identified a unique 57-amino acid (Bap1-57aa) sequence as the primary contributor to V. cholerae adhesion on various abiotic surfaces and on lipid membranes. However, the molecular mechanism underlying how this sequence binds to lipids remains unknown. In Chapter 4, we established multiple in vitro characterization methods to quantitatively assess the adsorption affinity of the Bap1-57aa peptide and to reveal the underlying molecular mechanisms of adhesion. Molecular dynamics simulations coupled with a fluorescence-based microbead adsorption assay revealed a central segment enriched in aromatic residues as the key driver of lipid adhesion. Synergistically, peripheral repeating units were shown to enhance lipid binding through avidity effects. Results from circular dichroism and infrared spectroscopy suggested that the central segment adopts a context-dependent -hairpin conformation to insert into lipid bilayers. By designing and testing peptide variants of different lengths and sequences and the corresponding V. cholerae mutants, we elucidated a detailed model for the Bap1-57aa peptide adhesion mechanism, with potential applications in targeted biofilm-removal strategies and biomaterial development for underwater glues. Overall, our findings reveal how Vibrio cholerae biofilms utilize modular adhesins with specialized roles to achieve robust adhesion across diverse surfaces. Additionally, we uncover the molecular mechanism by which the Bap1-57aa peptide interacts with lipid membranes, paving the way for future applications.

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