Protective and Anti-inflammatory Effects of Protegrin-1 on Citrobacter rodentium Intestinal Infection in Mice By Celina N. Osakowicz A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Animal Biosciences Guelph, Ontario, Canada © Celina N. Osakowicz, May, 2018 ABSTRACT PROTECTIVE AND ANTI-INFLAMMATORY EFFECTS OF PROTEGRIN-1 ON Citrobacter rodentium INTESTINAL INFECTION IN MICE Celina N. Osakowicz Advisor: University of Guelph, 2018 Dr. Julang Li Intestinal disorders and colitis affect millions of humans and food-animals world-wide. Antimicrobial peptides (AMPs) and their broad-spectrum antimicrobial activity represent a valuable potential therapeutic solution. Specifically, the potent pig-originated protegrin-1 (PG-1) has previously been shown to reduce the pathological effects of chemically induced digestive tract inflammation (colitis) along with modulation of immune responses and tissue-repair. This study aimed to extend these findings by investigating the potential protective effects of PG-1 on pathogen-induced intestinal colitis. We found that oral administration of PG-1 reduced Citrobacter rodentium intestinal infection in mice evidenced by; reduced histopathologic change in the colon, prevention of body weight loss, milder clinical signs of disease, and ultimately more effective clearance of bacterial infection relative to challenged mice. Additionally, PG-1 treatment altered the expression of various inflammatory mediators during infection to resolve inflammation and re-establish intestinal homeostasis. Interestingly, PG-1 administered in its mature form was most effective relative to the pro-form (ProPG-1). Acknowledgements I would like to acknowledge a great number of individuals who have been invaluable to this research project and whom the completion of this degree would not be possible without their support. First, I would like to express my utmost appreciation towards my advisor, Dr. Julang Li, for her countless support, guidance, and encouragement as my mentor throughout this stage of my academic career. During my masters, she always challenged me to do my best and supported my pursuit of knowledge and learning. I am truly grateful for having had the opportunity to work under such a passionate and dedicated researcher. I would like to thank my committee members, Dr. Jeff Caswell and Dr. Robert Friendship, for their valuable input and advice towards improving the research project and thesis. Additional thanks to Dr. Jeff Caswell for his assistance and expertise in the histological components of this project. To the members of the Li lab - past and present - thank you for all your support, thoughtful discussions, and friendship that helped foster a supportive lab environment. A special thank you goes out to Dr. Evanna Huynh and Dr. Jenna Penney, for their time and effort in enhancing my scientific learning and skills, and to James Haskins for his encouragement in navigating through the ups and downs of graduate studies. I would like to express my thanks to the Central Animal Facility (CAF) and Isolation Facility staff for their research animal expertise, care, and assistance in all my animal trials. A special thank you goes out to Veronique Carson and our undergraduate student Erin Miehe for their help and patience during the long hours of tissue collection. To my amazing parents, family, and friends, thank you for believing in me and for always being a constant source of love and encouragement. I am truly privileged to have you all in my life. Lastly, I would like to thank my partner, Michael Osborn, who has been there from the very beginning. You always managed to turn my bad days into good ones. Thank you for seeing me through this chapter of my life. It has been such a rewarding and exciting experience, and I look forward to what the future holds. Thank you all. iii Table of Contents ABSTRACT ................................................................................................................................................. ii Acknowledgements .................................................................................................................................... iii Table of Contents ....................................................................................................................................... iv List of Tables .............................................................................................................................................. vi List of Figures ............................................................................................................................................ vii List of Abbreviations ............................................................................................................................... viii 1. Introduction ............................................................................................................................................. 1 2. Literature Review ................................................................................................................................... 2 2.1 Overview of AMPs .................................................................................................................... 2 2.1.1 AMPs and their Structure ................................................................................................. 2 2.1.2 Mechanisms of Action ...................................................................................................... 4 1. Bactericidal .................................................................................................................. 4 2. Immunomodulatory Mechanism .................................................................................. 5 2.1.3 Multifunctional Roles of AMPs ....................................................................................... 7 2.1.4 AMPs in IBD .................................................................................................................... 8 2.2 Major Families of AMPs ........................................................................................................... 9 2.2.1 Defensins ......................................................................................................................... 9 2.2.2 Cathelicidins ................................................................................................................... 10 1. Protegrins ................................................................................................................... 11 2.3 Protegrin-1 ............................................................................................................................... 11 2.3.1 Mechanism and Structure ............................................................................................... 11 2.3.2 In vivo Applications of PG-1 .......................................................................................... 13 2.4 Recombinant PG-1 Production and Expression in Pichia pastoris ......................................... 14 2.5 Citrobacter rodentium ............................................................................................................. 18 3. Rationale and Research Objectives ..................................................................................................... 19 4. Materials and Methods ......................................................................................................................... 20 4.1 Construction and Transformation of ProPG-1-pUC Shuttle Vector into E. coli DH5 ........... 20 4.2 Excision of ProPG-1 from pUC Shuttle Vector ....................................................................... 20 4.3 Ligation of ProPG-1 into pD915-GAP Vector ........................................................................ 21 4.4 Confirmation of Ligation into pD915-GAP Vector ................................................................. 21 4.5 Transformation into P. Pastoris by Electroporation ................................................................ 22 4.6 Transformation Screening and Selection ................................................................................. 22 4.7 Shake-Flask Fermentation ....................................................................................................... 23 4.8 Bioreactor Fermentation & Optimization of Recombinant ProPG-1 ....................................... 23 4.9 Protein Sample Preparation ...................................................................................................... 24 4.10 SDS-PAGE & Western Blot Analysis ................................................................................... 25 4.11 Ethics Statement..................................................................................................................... 25 4.12 Bacterial Preparation .............................................................................................................. 25 4.13 Animals & Induction of Colitis .............................................................................................. 26 iv 4.14 Measurement of Bacterial Load ............................................................................................. 26 4.15 Fecal Water Content .............................................................................................................. 26 4.16 Disease Activity Index Score ................................................................................................