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Abstract

Enteric bacterial pathogens are a major cause of gastrointestinal disease worldwide, yet the strategies they use to overcome host defenses and establish infection remain poorly defined. A key barrier to enteric pathogen infection is the intestinal mucus layer, which protects intestinal epithelial cells (IECs) against microbial colonization. This thesis aims to determine how attaching and effacing (A/E) pathogens (such as Citrobacter rodentium) overcome this barrier to infect host cells. To achieve this goal, I performed three types of studies. First, I used 3D intestinal organoids to reveal the role of TLR2 in controlling intestinal epithelial barrier function. Second, I established an air-liquid interface (ALI) colonoid system that produces a physiologically relevant mucus layer with in vivo-like composition and glycosylation. This model demonstrated the critical role of mucus in delaying or even preventing C. rodentium from infecting IECs, consistent with our previous in vivo findings. Lastly, using the ALI system, I demonstrated that pre-exposure of C. rodentium to sialic acid markedly enhances its ability to degrade ALI-derived colonic mucus, which was associated with the secretion of two serine protease autotransporters of the Enterobacteriaceae (SPATEs), Pic and EspC. While Pic was already recognized as a mucinase, this work identified EspC as a novel mucin-degrading enzyme, representing the first class I SPATE shown to possess mucinolytic activity. Heterologous expression and recombinant protein studies confirmed EspC’s mucinolytic activity toward ALI-derived mucus. In conclusion, my thesis has used different organoid systems to understand microbe-host interactions. Using 3D organoids allowed us to define the role of TLR2 in responding to bacterial lipoproteins and maintaining IEC barrier function. Most importantly, ALI-derived organoid cultures allow us to reveal EspC as an atypical mucinase critical to C. rodentium pathogenesis, illustrating the great potential of this system to help dissect mucus-dependent pathogen-host interactions. These findings not only advance our mechanistic understanding of pathogen-host dynamics but also point toward translational opportunities for strengthening the mucus barrier as well as targeting microbial proteases to reduce pathogen virulence.