Functional Genomics at the EHEC-Intestinal Interface: Mechanisms of Pathogenicity and the Host Response

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a human foodborne pathogen that infects the colon, leading to hemorrhagic colitis and the potentially fatal hemolytic uremic syndrome. This dissertation aims to address the host-pathogen relationship between EHEC and the colonic mucosa in vivo. We used functional genomics to examine both requirements for EHEC pathogenicity and the enteric transcriptional response to infection in infant rabbits, a physiologically relevant animal model that closely recapitulates human EHEC intestinal disease. We used transposon-insertion sequencing screens to define EHEC genes required for both growth in vitro and colonic colonization. A transposon mutant library of over 100,000 unique EHEC mutants was analyzed to identify mutants whose frequency was reduced in different conditions. Underrepresented mutants provided knowledge of the pathogen’s essential gene set; moreover, passage of the transposon library through the infant rabbit colon yielded insight into the EHEC loci required for optimal intestinal colonization (Chapter 2). We identified many new colonization-promoting genes, including the widely conserved inner membrane protein CvpA (Chapter 2). Functional characterization of CvpA revealed that it is a member of the EHEC bile resistance repertoire and is linked genetically to the σE extracytoplasmic stress response (Chapter 3). We also assessed how EHEC’s potent toxin, Shiga toxin (Stx), modifies the histopathologic and transcriptional response to infection in the colon. We found that Stx is required for severe, hemorrhage and extensive apoptosis in the colon. Cellular-compartment-specific transcriptomics revealed that Stx dramatically remodels the host transcriptome during infection, particularly in epithelial cells. Genes related to coagulation and immune signaling pathways were differentially expressed in the presence of Stx, revealing the importance of this virulence factor in shaping the host response to infection (Chapter 4.) Collectively, these findings deepen understanding of the biology at the EHEC-intestinal interface. As no treatment exists for EHEC infection aside from rehydration therapy, these data will be useful to inform future work on development of new therapeutics.