THE EXAMINATION OF THE PROTEASE INHIBITOR ECOTIN AND N-LINKED GLYCOSYLATION IN CAMPYLOBACTER SPECIES, AN INSIGHT INTO CELL PROTECTION IN THE PROTEASE RICH ENVIRONMENT OF THE HUMAN HOST by CODY LOWELL CHARLES THOMAS (Under the Direction of Christine Szymanski) ABSTRACT Protection from proteolysis is important for bacteria living in a competitive niche under attack by host defense mechanisms. Campylobacter species possess an N-linked protein glycosylation system, where an oligosaccharide is attached to asparagine residues or released as free oligosaccharide. The glycosylation of these proteins can stabilize proteins such as the multidrug efflux pump of C. jejuni that helps efflux antibiotics and toxic compounds. Glycosylation can also mask cleavage sites protecting against proteolytic activity. Oral Campylobacter species have also developed an additional method for protection against proteases. Ecotin homologues have been identified in these non-thermophilic Campylobacter species. Ecotin is a generic serine-protease inhibitor that can bind and inhibit a wide range of proteases released by the host. The combination of N-glycosylation and ecotin helps provide Campylobacter species inhabiting the protease-rich oral cavity with additional mechanisms for bacterial fitness and survival in the human host. INDEX WORDS: Campylobacter jejuni, Campylobacter rectus, Campylobacter showae, ecotin, N-glycosylation, free oligosaccharide, protease inhibition, neutrophil elastase THE EXAMINATION OF THE PROTEASE INHIBITOR ECOTIN AND N-LINKED GLYCOSYLATION IN CAMPYLOBACTER SPECIES, AN INSIGHT INTO CELL PROTECTION IN THE PROTEASE RICH ENVIRONMENT OF THE HUMAN HOST By CODY LOWELL CHARLES THOMAS B.S., University of Alberta, 2015 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2018 © 2018 Cody Lowell Charles Thomas All Rights Reserved The examination of the protease inhibitor ecotin and N-linked glycosylation in Campylobacter species, an insight into cell protection in the protease rich environment of the human host By Cody Lowell Charles Thomas Major Professor: Christine Szymanski Committee: Robert Maier Kelly Moremen Electronic Version Approved: Suzanne Barbour Dean of the graduate School The University of Georgia August 2018 Dedication This dissertation is dedicated first and foremost to myself. I never would have expected myself at this juncture of my life if I did not push myself. I also dedicate this to my parents: Lowell Thomas and Verene Thomas, for all their love, patience and wisdom growing up in their care. iv Acknowledgements I would like to express my sincere gratitude to Dr. Christine M. Szymanski for allowing me to conduct my thesis research in her laboratory. Allowing me to transfer with her to the University of Georgia to complete my research at the Complex Carbohydrate Research Center. This work would not be completed if not for her advice and guidance. She is one of the top in her field and I consider myself extremely privileged to have had worked with her and learn from her. I had a wonderful experience throughout my graduate degree. I would like to thank Dr. Harald Nothaft for being a mentor to me throughout my degree. Harald is a well of knowledge and experience that has taught me almost everything I know in the laboratory. Harald was always there to answer questions and help develop methods to answering questions. I have learnt so much from my mentor Dr. Nothaft. I would like to thank my co-supervisors, Dr. Robert Maier and Dr. Kelley Moremen for providing feedback and useful suggestions throughout the duration of my degree. I would like to thank other members of the Szymanski laboratory: Robert Patry, Clay Crippen, Corey Wenzel, Justin Duma, Silke Andresen, Jolene Garber, Jessica Sacher as well as other past and present members. I am thankful to have worked by all your sides in the laboratory. I would like to thank Dr. Balaz Rada and his laboratory. Collaboration with the Rada lab was a major part of my research. I enjoy every day I would have to go work with neutrophils in the Rada lab, everyone was always so friendly and helpful. Thank you to everyone for making my graduate studies at the University of Alberta and the University of Georgia so memorable and enjoyable. v Table of contents Acknowledgements ......................................................................................................................... v List of tables ................................................................................................................................. viii List of figures ................................................................................................................................. ix List of abbreviations ..................................................................................................................... xii Preface........................................................................................................................................... xv Chapter I.......................................................................................................................................... 1 1.1 Overview of protein glycosylation ........................................................................................ 1 1.2 Overview of O-linked glycosylation ..................................................................................... 3 1.3 Overview of N-linked protein glycosylation ......................................................................... 6 1.4 Oral Campylobacter species................................................................................................ 13 1.5 Serine proteases ................................................................................................................... 14 1.6 Ecotin: a periplasmic serine protease inhibitor ................................................................... 15 1.6.2 Ecotin in Pseudomonas aeruginosa ................................................................................. 18 1.7 Overview of neutrophil killing ............................................................................................ 19 1.8 Thesis objectives ................................................................................................................. 22 1.9 References ........................................................................................................................... 24 Chapter II ...................................................................................................................................... 39 2.1 Introduction ......................................................................................................................... 41 2.2 Materials and methods ........................................................................................................ 44 2.3 Results ................................................................................................................................. 51 2.4 Discussion ........................................................................................................................... 58 vi 2.5 References: .......................................................................................................................... 81 3.1 Introduction ......................................................................................................................... 85 3.2 Materials and methods ........................................................................................................ 89 3.3 Results ................................................................................................................................. 91 3.4 Discussion ......................................................................................................................... 100 3.5 References: ........................................................................................................................ 115 Chapter IV ................................................................................................................................... 121 4.1 Research purpose............................................................................................................... 121 4.2 Summary and future directions ......................................................................................... 121 4.3 Conclusions and perspectives ........................................................................................... 127 4.4 References: ........................................................................................................................ 129 5.1 Introduction ....................................................................................................................... 134 5.2 Results ............................................................................................................................... 137 5.3 Discussion ......................................................................................................................... 154 5.4 Materials and Methods ...................................................................................................... 161 5.5 Supplementary data ........................................................................................................... 169 5.6 References: .......................................................................................................................