Escherichia coli O157:H7 and lactoferrin modulate immunity in cattle Evelien Kieckens Laboratory of Immunology Department of Virology, Parasitology and Immunology Thesis is submitted in fulfillment of the requirement for the degree of Doctor in Veterinary Sciences (PhD), Faculty of Veterinary Medicine, Ghent University, 2016 "A model is a lie that helps you see the truth." Howard Skipper The author and promoters give the permission to consult and to copy parts of this work for personal use only. Any other use is subject to the Copyright Laws. Permission to reproduce any material in this work should be obtained from the author. Promoters: Prof. Dr. Eric Cox Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University Prof. Dr. Daisy Vanrompay Department of Animal Production, Faculty of Bioscience Engineering, Ghent University Prof. Dr. Lieven De Zutter Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University Members of the Examination Committee: Prof. Dr. Piet Deprez (Chairman of the Examination Committee) Department of Internal medicine and clinical biology of large animals Faculty of Veterinary Medicine, Ghent University, Belgium Prof. Dr. Herman Favoreel (Secretary of the Examination Committee) Department of Virology, Parasitology and Immunology Faculty of Veterinary Medicine, Ghent University, Belgium Dr. ir. Koen De Reu Instituut voor Landbouw en Visserijonderzoek (ILVO), Belgium Prof. Dr. ir. Stefaan De Smet Department of Animal production Faculty of Faculty of Bioscience Engineering, Ghent University, Belgium Prof. Dr. Christian Menge Friedrich-Loeffler-Institut Institute of Molecular Pathogenesis, Jena, Germany Table of Contents Table of Contents List of abbreviations ................................................................................................................ 9 Chapter I: Literature review ................................................................................................... 11 1. Introduction ............................................................................................................... 13 2. Shiga toxin-producing Escherichia coli ........................................................................... 14 2.1 Nomenclature and taxonomy of Shiga toxin-producing Escherichia coli .................... 14 2.2 Pathogenesis ........................................................................................................ 16 2.3 Shiga toxin classification, structure, and role in pathogenesis ................................. 16 2.4 Colonization by formation of A/E lesions ............................................................. 19 2.5 The pO157 plasmid .............................................................................................. 22 2.6 Effector molecules................................................................................................ 22 2.7 E. coli O157:H7 infection ...................................................................................... 29 2.8 Immune responses against EHEC ......................................................................... 33 2.9 Strategies to reduce E. coli O157:H7 colonization in ruminants ............................... 40 3. Lactoferrin .................................................................................................................. 44 3.1 Introduction ........................................................................................................ 44 3.2 Structure of LF .................................................................................................... 44 3.3 Natural occurrence of LF ..................................................................................... 45 3.4 Biological functions and applications of LF ........................................................... 46 3.5 Modulation of the immune system by LF .............................................................. 47 3.6 Effect of LF on E. coli O157:H7 ............................................................................ 49 Chapter II: Aims of the study ................................................................................................. 51 Chapter III: Experimental work: Immunosuppressive effects of E. coli O157:H7 ....................... 55 3.1 Abstract .................................................................................................................... 57 3.2 Introduction.............................................................................................................. 58 3.3 Materials and methods .............................................................................................. 60 3.4 Results ..................................................................................................................... 63 3.5 Discussion ................................................................................................................ 78 3.7 Conclusions .............................................................................................................. 80 Chapter IV: Experimental work: Modulation of systemic immune responses by bLF ............... 83 4.1 Abstract .................................................................................................................... 85 4.2. Introduction............................................................................................................. 85 4.3. Materials and methods ............................................................................................. 87 4.4. Results .................................................................................................................... 90 4.5 Discussion ................................................................................................................ 98 7 Table of Contents 4.6 Conclusions ............................................................................................................ 101 4.7 Acknowledgments................................................................................................... 101 Chapter V: Experimental work: Effect of Stx2/bLF on release of proinflammatory cytokines .. 103 5.1 Abstract .................................................................................................................. 105 5.2 Introduction............................................................................................................ 105 5.2 Materials and methods ............................................................................................ 107 5.3 Results ................................................................................................................... 111 5.4 Discussion .............................................................................................................. 116 5.5 Conclusions ............................................................................................................ 119 Chapter VI: Experimental work: Effect of bLF on release and bioactivity of Stx ..................... 121 6.1 Abstract .................................................................................................................. 123 6.2 Introduction............................................................................................................ 123 6.3 Methods ................................................................................................................. 125 6.4 Results ................................................................................................................... 129 6.5 Discussion .............................................................................................................. 139 6.6 Conclusions ............................................................................................................ 142 6.7 Acknowledgements ................................................................................................. 142 Chapter VII: General discussion & future perspectives .......................................................... 143 Summary ............................................................................................................................ 153 Summary ............................................................................................................................ 155 Samenvatting ...................................................................................................................... 159 Samenvatting ...................................................................................................................... 161 References ........................................................................................................................... 165 References ........................................................................................................................... 167 Curriculum Vitae & Bibliography ........................................................................................ 197 Curriculum Vitae ................................................................................................................. 199 Bibliography ........................................................................................................................ 200 Dankwoord ......................................................................................................................... 203 8 List of abbreviations List of abbreviations A/E lesions: attaching and