Faculty of Biosciences, Fisheries and Economics Norwegian College of Fishery Science Francisella noatunensis ssp. noatunensis in Atlantic cod - Intracellular localization, innate immune responses and antibacterial proteins — Kathrine Ryvold Bakkemo A dissertation for the degree of Philosophiae Doctor – September 2016 TABLE OF CONTENTS ABBREVIATIONS AND ACRONYMS ................................................................................. I ACKNOWLEDGEMENTS .................................................................................................... II SUMMARY ............................................................................................................................. IV SAMMENDRAG .................................................................................................................... VI LIST OF PAPERS ............................................................................................................... VIII PAPER I ........................................................................................................................................... VIII PAPER II ......................................................................................................................................... VIII PAPER III ........................................................................................................................................ VIII 1. INTRODUCTION ............................................................................................................ 1 ATLANTIC COD IN AQUACULTURE .................................................................................................... 1 Diseases in cod .............................................................................................................................................. 2 Francisellosis ................................................................................................................................................. 2 THE IMMUNE SYSTEM IN VERTEBRATES .......................................................................................... 4 Innate defence mechanisms ........................................................................................................................... 4 Pathogen recognition ................................................................................................................................ 5 Inflammation............................................................................................................................................. 7 Subcellular mediators of the innate immune system ................................................................................. 9 The adaptive immune system ...................................................................................................................... 12 Cod have a unique immune system ............................................................................................................. 13 Phagocytosis and macrophage defences against intracellular pathogens..................................................... 14 Phagocytosis ........................................................................................................................................... 15 Macrophages in teleosts .......................................................................................................................... 15 Activation of macrophages ..................................................................................................................... 16 Killing mechanisms by macrophages ..................................................................................................... 17 INTRACELLULAR SURVIVAL OF PATHOGENS – SLEEPING WITH THE ENEMY .............................. 19 Francisella tularensis .................................................................................................................................. 21 Overview of the life cycle of F. tularensis ............................................................................................. 21 Survival mechanisms of F. tularensis ..................................................................................................... 24 Francisella in aquatic organisms ................................................................................................................. 25 2. AIMS OF STUDY .......................................................................................................... 27 3. ABSTRACT OF PAPERS ............................................................................................. 28 PAPER I ............................................................................................................................................. 28 PAPER II ........................................................................................................................................... 29 PAPER III .......................................................................................................................................... 30 4. DISCUSSION ................................................................................................................. 31 ESTABLISHMENT OF A PROTOCOL FOR GENTAMICIN PROTECTION ASSAY ................................. 31 INTRACELLULAR LOCALIZATION IN COD MACROPHAGES ............................................................ 32 SURVIVAL AND REPLICATION OF F. NOATUNENSIS IN MACROPHAGES AND ACL-CELLS ........... 33 PHAGOCYTOSIS IS ACTIN DEPENDENT ............................................................................................ 35 RECOMBINANT IFN-Γ LIMITS INTRACELLULAR SURVIVAL OF F. NOATUNENSIS ........................ 36 INNATE IMMUNE RESPONSES IN MACROPHAGES ........................................................................... 36 Gene expression of cytokines in macrophages following F. noatunensis infection .................................... 37 Gene expression of cytokines in macrophages after LPS treatment ............................................................ 37 ANTIBACTERIAL PROTEINS IN COD ................................................................................................ 38 Cod lack chicken-type lysozyme ................................................................................................................. 38 Distribution of antibacterial peptides in cod organs and tissues .................................................................. 39 LPS from E. coli does not induce expression of g-type lysozymes ............................................................. 41 F. noatunesis induce gene expression of antibacterial peptides .................................................................. 41 In vivo ..................................................................................................................................................... 41 In vitro .................................................................................................................................................... 42 5. CONCLUSIONS ............................................................................................................. 44 6. FUTURE PERSPECTIVE ............................................................................................ 45 7. APPENDIX ..................................................................................................................... 46 8. REFERENCES ............................................................................................................... 48 PAPER I-III ABBREVIATIONS AND ACRONYMS AMP Antimicrobial peptide ACL-cells Atlantic cod larvae cells APC Antigen presenting cell CFU Colony forming unit CMI Cell-mediated immunity CR Complement receptor CTL Cytotoxic T lymphocyte DC Dendritic cell FcγR Fc (Fc = region of immunoglobulins) gamma receptor FPI Francisella pathogenicity island GAS IFN-gamma activation site IFN Interferon IL Interleukin IRF Interferon regulatory factor Janus Activated Kinase and JAK-STAT Signal Transducer and Activator of Transcription LPS Lipopolysaccharide MAC Membrane attack complex MHC Major histocompatibility complex MR Mannose receptor NADPH Nicotinamide adenine dinucleotide phosphate NF-κB Nuclear factor kappa B NK cells Natural killer cells NO Nitric oxide OMV Outer membrane vesicle PAMP Pathogen-associated molecular pattern PRR Pattern recognition receptor ROI Reactive oxygen intermediates SE-N Surface-exposed nucleolin SR-A Scavenger receptor class A TEM Transmission electron microscopy TH cell T helper cell TLR Toll-like receptor TNF Tumor necrosis factor TS-WGD Teleost-specific whole genome duplication i ACKNOWLEDGEMENTS It is my pleasure to give thanks to several of the people who have been involved in my PhD-work. First of all, I would like to express my gratitude to my supervisor and project leader of the doctoral project, Dr. Marit Seppola, for the opportunity I was given to conduct my thesis. Financial support was provided by The Research Council of Norway grant no. 199672/E40. The project soon turned out to be a much more handful at the lab than first expected, and I am sincerely grateful for the support and guidance that you provided in the time following and all your help with manuscripts, in addition to valuable comments of this thesis. Next I am particularly grateful to my main supervisor at the University, Professor Børre Robertsen. Thank you for considering me as your PhD-student and the highly valuable comments with respect