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Innate Mechanisms of Antimicrobial Defense Associated with the Avian Eggshell Megan Rose-Martel

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Innate Mechanisms of Antimicrobial Defense Associated with the Avian Eggshell Megan Rose-Martel Innate Mechanisms of Antimicrobial Defense Associated with the Avian Eggshell Megan Rose-Martel Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the Doctorate in Philosophy degree in Cellular and Molecular Medicine Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Megan Rose-Martel, Ottawa, Canada, 2015 Dedication This thesis is dedicated to the loving memory of my father. He was a man in constant pursuit of knowledge. I was constantly reminded of how proud he was of my accomplishments, both personal and scientific. He read every article I published, every poster I created and inquired constantly about the research I was conducting, wanting to know every detail. His unwavering support and encouragement was invaluable to the completion of this thesis. I am extremely saddened that he is not here with us to see the completion of this chapter of my life which would have never been possible without him. I also dedicate this thesis to my loving husband, my wonderful mother and my darling daughter for their endless love, patience and support that they show towards me every day of my life. They have helped me through the most difficult times by listening to me, making me laugh and always having faith that I could make it. They are and will always be a constant source of comfort, love and happiness. ii Acknowledgments I would like to express my sincere gratitude to my supervisor and mentor, Dr. Maxwell Hincke, for the opportunity to work in his laboratory. His encouragement, guidance and motivation throughout my Ph.D. studies have made this research possible. I would also like to thank the members of my thesis advisory committee, Dr. Simon Lemaire, Dr. Alain Stintzi and Dr. Franco Pagotto for their advice and insightful discussions. I would also like to thank all my labmates for making the duration of my studies so enjoyable. I am especially grateful to the friendship provided by Jennifer Major, with whom lively discussions about life and science have helped me maintain good mental health. I would like to thank Dr. Syed Sattar from the Centre for Research on Environmental Microbiology (CREM) for the generous donation of bacterial cultures which were crucial for this research. This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Poultry Industry Council (PIC) and the Agriculture and Agri-Food Canada (AAFC)–Canadian Poultry Research Council (CPRC) poultry cluster. iii Abstract During the course of evolution, the avian egg has developed multiple physical and chemical barriers in order to resist microbial challenges. These barriers are essential for the successful reproduction of avian species as well as to maintain safe and nutritious food for human consumption of the table egg. The calcified eggshell is a biomineralized barrier with an integrated organic matrix containing antimicrobial proteins, a hallmark of sophisticated biological structures. Calcium carbonate is deposited onto the outer shell membranes to form the calcified mammillary, palisade and vertical crystal layers; the final layer to be deposited is the outer eggshell cuticle. In this thesis, mass spectrometry-based technology was used to investigate the proteome of the outer cuticle, the mammillary cones and the shell membranes in order to gain insight into biomineralization and antimicrobial functions of the avian eggshell. Proteomics analysis of the eggshell cuticle revealed multiple antimicrobial proteins, supporting the hypothesis that the outermost cuticle layer is the first barrier against invading pathogens. The two most abundant cuticle proteins identified are similar to Kunitz-like protease inhibitor (ovocalyxin-25) and ovocalyxin-32. Multiple antimicrobial proteins were also revealed to be associated with the shell membrane fibres. Among the most abundant proteins were lysozyme C, avian β-defensin-11, ovotransferrin, ovocalyxin-36 and gallin. The biomineralized shell is also an important physical barrier against invading pathogens. Proteomics analysis of the mammillary cones, the initiation sites for shell calcification, revealed several candidate proteins involved in calcitic biomineralization. Promising candidates include nucleobindin-2 and SPARC, two calcium binding proteins previously shown to modulate mineralization. In-depth analysis of the comprehensive proteomes generated by this study revealed the presence of histones in the shell iv membranes, shell and cuticle compartments. Histones are cationic antimicrobial peptides, which are key molecules of the innate immune defense system of many species. This thesis reports the minimal inhibitory concentrations and minimal bactericidal concentrations of histones extracted from avian erythrocytes against Gram-positive, Gram-negative and antibiotic-resistant bacteria. Results suggest that the underlying antimicrobial mechanism is based on the interaction between histones and lipopolysaccharides / lipoteichoic acids, which are negatively charged components of bacterial cell membranes. Histones also inhibit the growth of Gram-positive biofilms; the minimal biofilm eradication concentrations were determined for S. aureus and methicillin- resistant S. aureus (MRSA). Sensitive proteomics analyses have provided great insight into the protein constituents of the eggshell matrix, with two primary roles in the innate immune defense of the egg: regulation of calcitic biomineralization and antimicrobial protection. Further research on these proteins and their functions can provide a new focus for selective breeding programs looking to enhance the egg’s natural defenses, or provide inspiration for alternatives to conventional antibiotics, such as the histones. v Table of Contents Dedication………………………………………………………………………………….. ii Acknowledgments…………………………………………………………………………. iii Abstract…………………………………………………………………………………….. iv List of Figures...……………………………………………………………………………. xiii List of Tables………………………………………………………………………………. xvi List of Abbreviations………………………………………………………………………. xviii General Introduction 1 1. Food-borne Illnesses Associated with Contaminated Eggs….………………………….. 1 2. Egg Formation…………………………………………………………………………... 5 3. Biomineralized Barrier………………………………………………………………….. 11 3.1. Eggshell Membranes………………………………………………………….. 11 3.2. Mineralized Eggshell………………………………………………………….. 12 3.3. The Cuticle……………………………………………………………………. 15 4. Cationic Antimicrobial Peptides………………………………………………………… 17 4.1. Avian β-Defensins…………………………………………………………….. 18 4.2. Histones……………………………………………………………………….. 19 5. Statement of Hypotheses and Objectives……………………………………………….. 20 6. Thesis Outline…………………………………………………………………………… 21 vi Part 1 Theme: “Proteomics Analysis Provides Insight into Integrated Defense Strategies Operating at Biomineralized Barriers” 23 Chapter 1 “Protein Constituents of the Eggshell: Eggshell-Specific Matrix Proteins.” Rose, MLH and Hincke MT (2009) Cellular and Molecular Life Sciences, 66(16):2707- 19 24 Abstract…………………………………………………………………………………..... 25 Introduction…………………………………………………..……………………………. 26 Overview of Eggshell Biosynthesis…………………………………………………..……. 26 Biochemistry of Eggshell Matrix Proteins………………………………………………… 30 Biomineralization………………………………………………………………….. 33 Antimicrobial Protection…………………………………..………………………. 34 Eggshell-Specific Matrix Proteins…………………………………………………………. 34 Ovocleidin-17……………………………………………………………………… 34 Ovocleidin-116…………………………………………………………………….. 41 Ovocalyxin-32……………………………………………………………………... 45 Ovocalyxin-36……………………………………………………………………... 48 Other Eggshell-Specific Proteins………………………………………………….. 49 Non-Avian Eggshell Matrix Proteins……………………………………………… 50 vii Conclusions / Perspectives………………………………………………………………… 51 References……………………………………………………………………………….… 53 Chapter 2 “Proteomic Analysis Provides New Insight into the Chicken Eggshell Cuticle.” Rose-Martel M, Du J and Hincke MT (2012) Journal of Proteomics, 75(9):2697-706 66 Abstract…………………………………………………………………………………….. 67 1. Introduction……………………………………………………………………………... 68 2. Materials and Methods………………………………………………………………….. 69 2.1. Cuticle Protein Extraction…………………………………………………….. 69 2.2. Protein In-Gel Digestion………………………………………………………. 70 2.3. Mass Spectrometry……………………………………………………………. 70 2.4. Database Searching……………………………………………………………. 71 2.5. Criteria for Protein Identification……………………………………………... 71 2.6. Bioinformatics Analysis………………………………………………………. 72 3. Results………………………………………………………………………………….. 72 3.1. Cuticle Extraction…………………………………………………………….. 72 3.2. Cuticle Proteome……………………………………………………………… 74 3.3. Classification of Cuticle Proteins…………………………………………….. 79 4. Discussion……………………………………………………………………………….. 80 4.1. Eggshell-Specific Proteins…………………………………………………….. 82 4.2. Egg White Proteins……………………………………………………………. 83 viii 4.3. Ubiquitous/Miscellaneous Proteins…………………………………………… 84 4.4. Function of Cuticle Constituents……………………………………………… 85 References………………………………………………………………………………..... 88 Supplementary Data……………………………………………………………………….. 94 Chapter 3 “Novel Identification of Matrix Proteins Involved in Calcitic Biomineralization” Rose-Martel M, Smiley S and Hincke MT (2015) Journal of Proteomics, 116:81-96 105 Abstract…………………………………………………………………………………….. 107 1. Introduction……………………………………………………………………………... 108 2. Materials and Methods………………………………………………………………….. 111 2.1. Shell Membrane Removal from Fertilized Eggs……………………………… 111 2.2. Shell Membrane Removal from Unfertilized Eggs…………………………… 112 2.3. Harvesting Mammillary Cone Proteins
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