University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2021-01-15 The effects of diketopiperazines on the virulence of Burkholderia cepacia complex species Jervis, Nicole Marie Jervis, N. M. (2021). The effects of diketopiperazines on the virulence of Burkholderia cepacia complex species (Unpublished master's thesis). University of Calgary, Calgary, AB. http://hdl.handle.net/1880/113002 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY The effects of diketopiperazines on the virulence of Burkholderia cepacia complex species by Nicole Marie Jervis A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA JANUARY, 2021 © Nicole Marie Jervis 2021 Abstract Recognized as a novel class of quorum sensing inhibitors (QSIs), 2,5-diketopiperazines (DKPs) are small, organic molecules that hold many important physiochemical properties which has led to recent inquiries into their effects towards limiting the pathogenicity of multi- drug resistant bacterial pathogens. Species within the Burkholderia cepacia complex (Bcc) are one such group of pathogens that require attention towards the development of alternative therapeutic strategies given their detrimental clinical outcomes, particularly in patients with cystic fibrosis. By targeting Bcc QS regulation, our data demonstrated DKPs, cyclo(-D-ala-val), cyclo(-pro-val), cyclo(-leu-pro), and cyclo(-phe-pro) to alter the production of extracellular virulence factors and to decrease the production of biofilm-associated factors which comprise the protective extracellular matrix of Bcc biofilms. Further analysis demonstrated these DKPs to also possess antibiotic potentiator activity, enhancing the antimicrobial activity of ceftazidime, meropenem, and tobramycin. Taken together, the data collected in this study offers an initial step towards understanding the potential role of DKPs in the development of alternative therapeutics. ii Acknowledgements Foremost, I would like to acknowledge my supervisor Dr. Douglas Storey. Thank you for all your careful guidance, helpful advice and unwavering support that you have provided me throughout the years. After taking me on as an eager undergraduate research student, I am grateful for your belief in my abilities to continue on in graduate studies where your kindness and mentorship has helped shaped me into the researcher I am today. I am especially thankful for the opportunity I had to attend conferences, work as a teaching assistant, and learn the art of backyard croquet. Above all, your patience and understanding in allowing me the freedom to navigate through my project while also offering your valuable knowledge and encouragement will always be appreciated. I would also like to thank my committee members Dr. J. Harrison and Dr. M. Parkins, as well as Dr. A. Savchenko for all of their guidance and dedication towards helping me improve as a scientist. To all the members of the Storey lab, I will always be grateful for the memorable times we shared, both in the lab brainstorming ideas for experiments, as well as outside the lab gift wrapping at the mall, attending concerts, and wandering unknown areas of campus in search of Pokémon. To the Harrison lab, thank you for including me as an unofficial member by generously offering lab space to carry out experiments and for all your help and advice when said experiments did not turn out. To my labmate Nabiha Mehina, your support and friendship has been immeasurable throughout our time together. From our first day as new graduate students collecting an abundance of free pens at orientation, to our final days of jamming to BTS while scrambling to finish our theses, I owe you my deepest debt of gratitude that will no doubt carry on as we continue forward into the next chapters of our lives. To my partner, Daniel Aschauer, for everything you have done unconditionally to take care of my happiness and to support me throughout my studies, thank you. I am grateful to iii have shared these experiences with you and will forever hold them close as we continue to share more together in the future. Above all, I would like to thank my family for all their love and constant support in my academic endeavours. To my parents, Jennifer and Rocky, your encouragement to take on new challenges never ceases to motivate me to do better and I will eternally be grateful for your care in my well-being. To my sister, Vanessa, your energy and passion for life will always be a true inspiration that I will forever admire, thank you for sharing your light with me every day. iv Dedication To my parents, who provided me with the ability to find my own way. v Table of Contents Abstract ....................................................................................................................................................... ii Acknowledgements ............................................................................................................................... iii Dedication .................................................................................................................................................. v Table of Contents ................................................................................................................................... vi List of Tables ............................................................................................................................................ ix List of Figures and Illustrations......................................................................................................... x List of Symbols, Abbreviations and Nomenclature ................................................................... xi CHAPTER ONE: INTRODUCTION ...................................................................................................... 1 1.1 2,5-Diketopiperazines (DKPs) ............................................................................................... 1 1.1.1 Physiochemical properties ............................................................................................ 1 1.1.2 Biological significance ..................................................................................................... 3 1.1.3 DKPs as quorum sensing inhibitors ........................................................................... 4 1.1.4 DKPs of interest ................................................................................................................. 5 1.2 The Burkholderia cepacia complex (Bcc)........................................................................... 6 1.2.1 An important group of cystic fibrosis pathogens ................................................. 6 1.2.2 Pathogenic traits ............................................................................................................... 8 1.2.2.1 Extracellular virulence factors ......................................................................... 8 1.2.2.2 Biofilm formation ............................................................................................... 10 1.2.3 Quorum sensing regulation ....................................................................................... 14 1.2.4 Treatment of Bcc infections ....................................................................................... 16 1.3 Project Overview ...................................................................................................................... 19 1.3.1 Hypothesis ........................................................................................................................ 19 1.3.2 Objectives .......................................................................................................................... 19 CHAPTER TWO: MATERIALS AND METHODS.......................................................................... 20 2.1 Bacterial Strains and Growth Conditions ....................................................................... 20 2.1.1 Maintenance and growth ............................................................................................ 20 2.1.2 Synthetic cystic fibrosis sputum media ................................................................. 20 2.2 Preparation of 2,5-Diketopiperazines ............................................................................. 22 2.3 Growth Assessments .............................................................................................................. 24 2.3.1 Growth curves ................................................................................................................. 24 2.3.2 Viable cell enumeration ............................................................................................... 24 2.4 Extracellular Virulence Factor Assessments ................................................................. 25 2.4.1 Isolation of cell-free spent media ............................................................................ 25 2.4.2 Protease production ....................................................................................................