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Modulation of Listeria Monocytogenes Biofilm Formation Using Small Molecules and Enzymes

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Modulation of Listeria Monocytogenes Biofilm Formation Using Small Molecules and Enzymes MODULATION OF LISTERIA MONOCYTOGENES BIOFILM FORMATION USING SMALL MOLECULES AND ENZYMES MODULATION OF LISTERIA MONOCYTOGENES BIOFILM FORMATION USING SMALL MOLECULES AND ENZYMES By UYEN THI TO NGUYEN, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Uyen T.T. Nguyen, July 2014 Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences McMaster University DOCTOR OF PHILOSOPHY (2014) Hamilton, Ontario (Biochemistry and Biomedical Sciences) TITLE: Modulation of Listeria monocytogenes biofilm formation using small molecules and enzymes AUTHOR: Uyen Thi To Nguyen, B.Sc. (McMaster University) SUPERVISOR: Dr. Lori L. Burrows NUMBER OF PAGES: xvii, 217 ii Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences ABSTRACT Inadequately disinfected food contact surfaces colonized by Listeria monocytogenes can come into contact with ready-to-eat food products causing cross-contamination and food-borne outbreaks. L. monocytogenes is tolerant of high salt, low temperatures and low pH, in part due to its ability to form biofilms, defined as communities of microorganisms that are surrounded by a self-produced extracellular polymeric substance that can adhere to surfaces. Biofilm formation is a complex process involving a series of poorly defined physiological changes that together lead to tolerance of disinfectants and antibiotics. To better understand the process of L. monocytogenes biofilm development, and to investigate ways in which colonization of surfaces might be prevented, we developed a microtiter biofilm assay suitable for high throughput screening. The assay was used to identify small molecules (protein kinase inhibitors and previously FDA-approved bioactive drugs) that modulate L. monocytogenes biofilm development. Of the subset of molecules with biofilm modulatory activities, we showed that select protein kinase inhibitors and β-lactams prevented or reduced biofilm formation (<50% of vehicle control) of L. monocytogenes lab strains and food isolates at micromolar concentrations. In other cases, specific β-lactams stimulated biofilm formation (>200% of vehicle control) at sub-minimal inhibitory concentrations. Characterization of the penicillin-binding protein iii Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences targets of the β-lactams revealed that PBPD1, a low molecular weight D,D- carboxypeptidase, is targeted by non-stimulatory β-lactams. Stimulatory β- lactams did not increase biofilm formation of a pbpD1 mutant to the same extent as wild type. In addition to inhibiting biofilm formation, many β- lactams dispersed established biofilms, although not completely. However, targeting components of the EPS matrix with enzymes — specifically proteins, using proteinase K –completely blocked adhesion and removed established biofilms. Together, these findings demonstrate how molecules with different mechanisms of action can modulate biofilm formation and the potential for use of proteinase K in the food industry as an antibiofilm agent. iv Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Dr. Lori Burrows for her unconditional support and guidance throughout my years in graduate school. We had many obstacles experimenting on a bacterium that no one else in the Burrows lab works on, but we always managed to come out on top. Thank you for pushing me beyond my limits, always telling me to be many steps ahead of where I am, and look at the bigger picture. I am very thankful for your mentorship and encouragement on this journey. I would also like to thank my committee members, Dr. Gerard Wright, Dr. Justin Nodwell, and Dr. Michael Surette for their many experimental ideas, challenging questions and pushing me to read more and expand my knowledge. The amazing past and current members of the Burrows Lab, thank you for being part of my journey. Hanjeong, I would have never reached this stage without your love and support in the lab, thank you for all your help on this project. Ylan and Sara, thank you for always being there and for our many adventures both inside and outside the lab. To my biofilm partner Iwona, your support at the beginning of my experience will never be forgotten along with our travelling adventures. Dr. Ryan Lamers, Ryan, Tiffany, Vivian and Joseph, thank you for the laughter and help throughout the years. v Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences This experience would not have been complete without the love and strong support of my family. Thank you to my parents for their encouragement and always believing in me. To my siblings – Quyen, Julie, and Michael – thank you for the tears, laughter, love, and support. Lastly, to Angelo, you were there at the very beginning of my graduate studies and have witnessed the best and worst of it all. Thank you mahal ko for your love and words of encouragement to help me achieve where I am today. vi Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences TABLE OF CONTENTS ABSTRACT ............................................................................................... iii ACKNOWLEDGEMENTS .......................................................................... v TABLE OF CONTENTS ........................................................................... vii LIST OF FIGURES .................................................................................... xi LIST OF TABLES .................................................................................... xiii LIST OF ABBREVIATIONS .................................................................... xiv CHAPTER ONE - Introduction ................................................................. 1 Introduction .......................................................................................... 2 Listeria monocytogenes ....................................................................... 2 Listeriosis ............................................................................................. 4 Biofilm formation .................................................................................. 6 Biofilm prevention and removal ......................................................... 10 Identification of genes involved in L. monocytogenes biofilm formation ............................................................................................ 12 Mechanisms of biofilm resistance ...................................................... 13 EPS matrix limits diffusion of antimicrobial compounds ................. 13 Decreased metabolic activity to prevent killing by antimicrobial agents ....................................................................... 14 Increased resistance through activation of biofilm-specific genes ............................................................................................. 14 Tolerance to antimicrobial agents via dormancy ............................ 15 The cell surface of Listeria ................................................................. 16 The Listeria peptidoglycan layer .................................................... 17 The effects of β-lactams on PBP function ...................................... 19 Identification of L. monocytogenes PBPs ...................................... 20 The roles of autolysins in peptidoglycan breakdown and biofilm formation ........................................................................................ 22 Surface molecules: Incorporation into the cell wall and role in biofilm formation ............................................................................. 23 Two component systems of Listeria .................................................. 25 Purpose and research aims ............................................................... 31 CHAPTER TWO – Small-molecule modulators of Listeria monocytogenes biofilm development ............................................... 34 Co-authorship statement ................................................................... 35 Title page and author list ................................................................... 36 Abstract ............................................................................................. 37 Introduction ........................................................................................ 38 Materials and Methods ...................................................................... 42 Bacterial strains and culture conditions ......................................... 42 vii Ph.D. – U.T.T. Nguyen; McMaster University – Biochemistry and Biomedical Sciences Preparation of test compounds ...................................................... 43 Determination of minimal inhibitory concentrations (MICs) ............ 43 L. monocytogenes and S. aureus biofilm assays ........................... 44 Scanning electron microscopy of L. monocytogenes 568 on stainless steel ................................................................................ 46 Synthesis of acyl carnitines ............................................................ 47 LC-ESI-MS analysis ....................................................................... 48 1D and 2D NMR ............................................................................. 50 Compound Data ............................................................................
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