Role of Actinobacillus suis adhesins in host-pathogen interactions in the tonsils of the soft palate of swine by Adina Rachel Bujold A thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Doctor of Philosophy in Pathobiology Guelph, Ontario, Canada © Adina Rachel Bujold, May, 2016 ABSTRACT ROLE OF ACTINOBACILLUS SUIS ADHESINS IN HOST-PATHOGEN INTERACTIONS IN THE TONSILS OF THE SOFT PALATE OF SWINE Adina Rachel Bujold Advisor University of Guelph, 2016 Dr. Janet I. MacInnes Tonsils are the immune system’s first line of defence against antigens entering the upper respiratory tract. Paradoxically, they are also an important site of colonisation and can be a portal of entry for many pathogens. Despite their significant role, virtually nothing is known about how bacteria attach to or the factors that control invasion of tonsils. To address these questions, I have used the gram-negative facultative anaerobe, Actinobacillus suis, as a model. A. suis is a common resident of swine tonsils, but it sometimes invades the bloodstream, leading to systemic disease and death. Using a combination of bioinformatic and expression studies, I have shown that A. suis expresses different adhesins in growth conditions that mimic the environment of the tonsils and the biofilm and planktonic stages of the bacterium that might lead to host invasion. Three knockout mutants were created to better understand mechanisms of attachment of A. suis to tonsils, and experiments to identify host molecules involved in infection were completed. I found that disrupting adhesins involved in attachment and cell integrity (ompA), attachment to host connective tissues (comE1), and biofilm formation (flp1) resulted in reduced binding of A. suis to purified extracellular matrix components (fibronectin, vitronectin, laminin, collagen I, collagen IV), as well as reduced its ability to form biofilms. Together, these studies provide insight into the pathogenesis of A. suis and other important invasive pathogens known to colonise the tonsils of swine and other species. Acknowledgments First and foremost, I would like to thank my advisor, Dr. Janet MacInnes, for her supervision. I have enjoyed the relationship we have developed during the time we have worked together, and I appreciate the ways you have encouraged and challenged me. Thank you for your mentorship and friendship. I would also like to thank my advisory committee members: Drs. Patrick Boerlin, Marc Coppolino, and Brandon Lillie. You have each provided invaluable expertise, advice, and encouragement over the course of my program, and I am grateful for all the contributions you have made. I would also like to thank my labmates, past and present, from the MacInnes, Prescott, and Boerlin research groups. Thank you for your friendship and support throughout my program. Thank you to the collaborators I have had the pleasure of working with: Drs. Mario Jacques, Andrew Kropinski, and John Nash. Thank you to Jing Zhang and Dyanne Brewer from the Advanced Analytics Centre, and Jonathan Krieger at the SPARC Biocentre, for your technical expertise and assistance. I would like to thank the financial supporters of my program: the Ontario Veterinary College Scholarship program; the Ontario Ministry of Training, Colleges and Universities Ontario Graduate Scholarship program; Vétoquinol Canada Inc.; the department of Pathobiology; and the Korean-Canadian Scholarship Foundation. I am thankful for the financial support awarded to Dr. Janet MacInnes that has also funded my research: the Natural Sciences and Engineering Research Council and the Ontario Ministry of Agriculture, Food, and Rural Affairs. iv Last but not least, I would like to thank my family and friends for their endless support. In particular, thank you to my parents for their love and encouragement through all the successes and struggles in my life. Thank you to my strong and resilient sisters for being my friends and role models since my first day. Thank you to my Uncle Harold and Auntie Heather, for their love and enthusiastic encouragement of everything I do. And thank you to the old and new members of my family, who continue to enrich my life in so many ways. Also, thank you to Dr. Brenna Brown, who supported me during my program, and allowed me the pleasure of doing the same during hers. And to all the other friends who have celebrated and commiserated with me during this chapter in my life, I am truly grateful. Finally, to Dr. Anne Laarman: thank you for your partnership, your companionship, your friendship, and your love. I am so honoured to share my life, and now the title of Doctor, with you. v Dedication To my parents, Paul and Cynthia Bujold: Mom, you have been unwaiveringly behind me, at times cheering me on, at times holding me up, and often both in equal measure. The high personal bar that you set, your passion, and your selflessness inspire me so much. Thank you for being my first and most devoted cheerleader. Dad, you have modelled the attributes of a scholar and person that I strive every day to possess: a fair and measured assessment of the facts, an unquenchable curiosity for knowledge, and the integrity to stand up and speak truth under all circumstances. Your calm demeanour, dry wit, and humour have grounded me and buoyed me, whichever was required at the time. Also, I suspect you set me on this path at the age of four, when we lay on the floor reading a Wrinkle in Time and learned about folding time. Now that my time has converged on this particular point, I share this accomplishment with you. To my partner and best friend, Anne Laarman: I don’t know where to begin. I admire and look up to you in so many ways I can’t even enumerate. You inspire me, you ground me, you support me, and you love me. Thank you for sharing the load and walking beside me, now and always. vi Declaration of Work Done I declare that this thesis, entitled “Role of Actinobacillus suis adhesins in host-pathogen interactions in the tonsils of the soft palate of swine”, is entirely my own work and that where any material could be construed as the work of others, it is fully cited and referenced, with appropriate acknowledgement given. All experiments were designed and executed solely by me, with the following exceptions: screening additional A. suis isolates for the presence of adhesin genes by real-time PCR, where technical assistance was provided by Glenn Soltes; generating A. suis isogenic mutants, where technical assistance was provided by Sarah Walker; conducting biofilm assays, where technical assistance was provided by Josée Labrie; and preparing tonsil samples for mass spectrometry, where technical assistance was provided by Kearin Devlin. Signature: Name of Student: Adina R. Bujold Name of advisor: Janet I. MacInnes vii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Dedication ...................................................................................................................................... vi Declaration of Work Done ............................................................................................................ vii Table of Contents ......................................................................................................................... viii List of Tables ............................................................................................................................... xiv List of Figures .............................................................................................................................. xvi List of Symbols and Abbreviations............................................................................................ xviii Chapter 1: Literature Review .......................................................................................................... 1 1.1 Preamble ........................................................................................................................... 1 1.2 Tonsils .............................................................................................................................. 1 1.2.1 Types of tonsils ......................................................................................................... 1 1.2.2 Anatomy .................................................................................................................... 2 1.2.3 Histology ................................................................................................................... 4 1.2.3.1 Epithelium ............................................................................................................. 4 1.2.3.2 Leucocyte populations........................................................................................... 7 viii 1.2.4 Role in immunity ...................................................................................................... 9 1.2.4.1 Innate immunity .................................................................................................... 9 1.2.4.2 Adaptive immunity .............................................................................................. 11 1.2.5 Role in disease .......................................................................................................