Author ORCID Identifier

0000-0003-0488-7473

Date of Graduation

12-2023

Document Type

Dissertation (PhD)

Program Affiliation

Microbiology and Infectious Diseases

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Anne-Marie Krachler, Ph.D.

Committee Member

George Eisenhoffer, Ph.D.

Committee Member

Michael Lorenz, Ph.D.

Committee Member

Anna Konovalova, Ph.D.

Committee Member

Michael Galko, Ph.D.

Abstract

The dynamic nature and inaccessible location of the intestine pose significant challenges to the study of intestinal physiology and pathology. Zebrafish larvae, possessing optical transparency and genetic tractability, offer an accessible and clinically relevant model for investigating dynamic events in the intestine via time-lapse imaging. In the first part of this work, I discuss our efforts to optimize the parameters of a foodborne infection assay using paramecia as a vehicle. This method provides an effective, high-throughput alternative to infection via immersion or oral gavage, and replicates the most common route of transmission of gastrointestinal (GI) infection in humans. The foodborne infection assay was then used in combination with enteric nervous system (ENS)-deficient zebrafish, a model for Hirschsprung’s disease (HSCR), to study the effects of the ENS on the colonization and virulence gene expression of enterohemorrhagic Escherichia coli (EHEC). My findings suggest that a functional ENS plays a crucial role in shaping intestinal disease outcomes.

The intestinal epithelium, a critical barrier against pathogens, toxins, and other environmental stressors, undergoes rapid turnover. Extrusion, the process by which excess or compromised cells are shed from epithelial layers, is vital to maintaining barrier integrity. Despite extensive research in various models, including tissue culture cells, Drosophila, and the tail fin epithelia of zebrafish larvae, a model to study intestinal epithelial extrusion in vivo is lacking. In the second part of my dissertation, I address this gap by generating a novel transgenic zebrafish line that permits a time resolved analysis of intestinal epithelial extrusion. Using this model in tandem with a fluorescent reporter for neutrophils, I demonstrate regional variations in neutrophil recruitment and dynamics following intestinal cell extrusion. These results emphasize the role of intestinal homeostasis in the regulation of immune responses. Overall, these studies have generated new tools with potential applications in host-microbe interactions and barrier dysfunction diseases.

Keywords

larval zebrafish, enterohemorrhagic E. coli, epithelial extrusion, intestinal damage, enteric nervous system

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