Diarrheagenic Escherichia coli signaling and interactions with host innate immunity and intestinal microbiota by Gaochan Wang B.S., China Agricultural University, 2007 M.S., The Ohio State University, 2012 AN ABSTRACT OF A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2017 Abstract Diarrheagenic Escherichia coli (E. coli) strains are common etiological agents of diarrhea. Diarrheagenic E. coli are classified into enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC or enterohemorrhagic E. coli [EHEC]), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and adherent invasive E. coli (AIEC). In addition to encoding toxins that cause diarrhea, diarrheagenic E. coli have evolved numerous strategies to interfere with host defenses. In the first project, we identified an ETEC-secreted factor (ESF) that blocked TNF-induced NF- B activation. One of the consequences of TNF-induced NF-B activation is the production of pro-inflammatory cytokines that help to eliminate pathogens. Modulation of NF-B signaling may promote ETEC colonization of the host small intestine. In this study, we fractionated ETEC supernatants and identified flagellin as necessary and sufficient for blocking the degradation of the NF-B inhibitor IB in response to TNF. In the second project, we attempted to identify an ETEC cAMP importer. ETEC diarrhea leads to cAMP release into the lumen of the small intestine. cAMP is a key secondary messenger that regulates ETEC adhesin expression. We hypothesized that a cAMP importer is present in ETEC, accounting for its hypersensitivity to extracellular cAMP. We used Tn5 transposome-mediated mutagenesis to construct a mutant library and screen for cAMP-hyporesponsive mutants. However, none of the 17,956 mutants we screened were cAMP-hyporesponsive. In the third project, we focused on gut microbiota and the T3SS effector NleH. We used the mouse-specific pathogen C. rodentium and transplanted performed microbiota between different mouse strains. We evaluated microbiota populations as a function of infection with WT and nleH C. rodentium strains before and after microbiota transplantation. Microbiota transfer altered the resistance to WT C. rodentium infection in C57BL/10ScNJ mice and the NleH effector promoted host resistance to C. rodentium. Diarrheagenic Escherichia coli signaling and interactions with host innate immunity and intestinal microbiota by Gaochan Wang B.S., China Agricultural University, 2007 M.S., The Ohio State University, 2012 A DISSERTATION submitted in partial fulfillment of the requirements for the degree DOCTOR OF PHILOSOPHY Department of Diagnostic Medicine/Pathobiology College of Veterinary Medicine KANSAS STATE UNIVERSITY Manhattan, Kansas 2017 Approved by: Major Professor: Dr. Philip Hardwidge Copyright © Gaochan Wang 2017. Abstract Diarrheagenic Escherichia coli (E. coli) strains are common etiological agents of diarrhea. Diarrheagenic E. coli are classified into enterotoxigenic E. coli (ETEC), Shiga toxin-producing E. coli (STEC or enterohemorrhagic E. coli [EHEC]), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffuse-adherent E. coli (DAEC), and adherent invasive E. coli (AIEC). In addition to encoding toxins that cause diarrhea, diarrheagenic E. coli have evolved numerous strategies to interfere with host defenses. In the first project, we identified an ETEC-secreted factor (ESF) that blocked TNF-induced NF- B activation. One of the consequences of TNF-induced NF-B activation is the production of pro-inflammatory cytokines that help to eliminate pathogens. Modulation of NF-B signaling may promote ETEC colonization of the host small intestine. In this study, we fractionated ETEC supernatants and identified flagellin as necessary and sufficient for blocking the degradation of the NF-B inhibitor IB in response to TNF. In the second project, we attempted to identify an ETEC cAMP importer. ETEC diarrhea leads to cAMP release into the lumen of the small intestine. cAMP is a key secondary messenger that regulates ETEC adhesin expression. We hypothesized that a cAMP importer is present in ETEC, accounting for its hypersensitivity to extracellular cAMP. We used Tn5 transposome-mediated mutagenesis to construct a mutant library and screen for cAMP-hyporesponsive mutants. However, none of the 17,956 mutants we screened were cAMP-hyporesponsive. In the third project, we focused on gut microbiota and the T3SS effector NleH. We used the mouse- specific pathogen C. rodentium and transplanted performed microbiota between different mouse strains. We evaluated microbiota populations as a function of infection with WT and nleH C. rodentium strains before and after microbiota transplantation. Microbiota transfer altered the resistance to WT C. rodentium infection in C57BL/10ScNJ mice and the NleH effector promoted host resistance to C. rodentium. Table of Contents List of Figures ................................................................................................................................ xi List of Tables ............................................................................................................................... xiii Acknowledgements ...................................................................................................................... xiv Chapter 1 - Literature Review......................................................................................................... 1 Escherichia coli .......................................................................................................................... 1 Pathogenic of Escherichia coli ................................................................................................... 1 Classification of Escherichia coli ............................................................................................... 2 Serotypes of Escherichia coli ................................................................................................. 2 O-antigens ........................................................................................................................... 2 H-antigens ........................................................................................................................... 2 K-antigens ........................................................................................................................... 3 Phylotypes of Escherichia coli ............................................................................................... 3 Pathotypes of Escherichia coli ................................................................................................ 4 Enterotoxigenic Escherichia coli ........................................................................................ 5 Shiga toxin-producing Escherichia coli.............................................................................. 7 Enteropathogenic Escherichia coli ..................................................................................... 8 Enteroaggregative Escherichia coli .................................................................................... 9 viii Enteroinvasive Escherichia coli ....................................................................................... 10 Diffuse-adherent Escherichia coli .................................................................................... 11 Adherent Invasive Escherichia coli .................................................................................. 12 Innate immunity and pathogen recognition .............................................................................. 13 Toll-like receptors ................................................................................................................. 15 NOD-like receptors ............................................................................................................... 18 NF-B signaling pathway ......................................................................................................... 20 TNF-NF-B signaling pathway ................................................................................................ 23 Gut microbiota and enteric pathogens ...................................................................................... 26 Cyclic adenosine monophosphate (cAMP) and cAMP-receptor protein (CRP) in Escherichia coli ............................................................................................................................................ 30 Modulation of inflammatory responses .................................................................................... 34 Colonization of pathogenic Escherichia coli in hosts .............................................................. 36 Chapter 2 - Enterotoxigenic Escherichia coli flagellin blocks TNF-induced NF-B activation . 43 Introduction ............................................................................................................................... 43 Materials and Methods.............................................................................................................. 45 Results ....................................................................................................................................... 50