Identification and Characterization of Proteolytic Activities from S. mutans that Hydrolyzes Dentinal Collagen Matrix Bo Huang, DMD, PhD A thesis submitted in conformity with the requirements for the degree of MSc Faculty of Dentistry University of Toronto © Copyright by Bo Huang (2021) Identification and Characterization of Proteolytic Activities from S. mutans that Hydrolyzes Dentinal Collagen Matrix A thesis submitted in conformity with the requirements for the degree of MSc (2021) Bo Huang, DMD, PhD Faculty of Dentistry University of Toronto Abstract Objective: To measure the proteolytic activity of S. mutans, its discrete fractions, and proteases towards demineralized human dentin. Methods: Demineralized human dentin slabs were incubated with either medium, cultures (overnight or newly inoculated) of S. mutans UA159, or different bacterial fractions (intracellular, supernatant or bacterial membrane). Media from each condition was analyzed for a collagen degradation marker, hydroxyproline. Three potential proteolytic enzymes (SMU_759, SMU_761 and SMU_1438c) from S. mutans UA159 were expressed and their activity toward dentinal collagen was measured based on hydroxyproline analysis. Results: Media only and bacterial membrane had no activity towards dentinal collagen. Overnight culture of S. mutans had the highest degradative activity (p<0.05), followed by supernatant and intracellular component, and newly inoculated culture (p<0.05). SMU_759 had the highest degradative activity towards dentinal collagen, followed by SMU_761 (p<0.05). SMU_1438c showed no collagen degradative activity (p<0.05). Conclusion: S. mutans dentinal collagen degradation could potentially contribute to caries formation. ii Acknowledgments I would like to thank my supervisors, Dr. Yoav Finer and Dr. Dennis Cvitkovitch, for giving me this opportunity to work with them. They have been excellent mentors. This thesis would not have been possible without their support. I would also like to thank my committee members, Dr. Christopher McCulloch and Dr. Paul Santerre, whose insights and suggestions helped me to improve the quality of this project. I have been lucky to work with great people in the Dr. Finer’s laboratory, who have created a great work environment: Dr. Cameron Stewart, Russel Gitalis, Dr. Ousama Damlaj. I would also like to thank members in Dr. McCulloch’s laboratory. I am also very grateful to my family for their support throughout this process, in particular my husband Liang Ren, who has always been there for me, encourages me, guides me and understands me. And, thank you, my lovely children, Claire and Ajax, for your hugs, kisses, smiles and kind letters. At the last, but not the least, I would love to express my sense of gratitude to my sister, Youning, who has been so caring and supportive during the 3 years. iii Table of Contents Abstract ......................................................................................................................................... II List of tables................................................................................................................................. VI List of figures ............................................................................................................................. VII List of abbreviations ................................................................................................................ VIII Preface .......................................................................................................................................... IX Chapter 1 introduction ................................................................................................................. 1 1.1 INTRODUCTION ..................................................................................................................... 1 1.2 HYPOTHESES ....................................................................................................................... 3 1.3 OBJECTIVES ........................................................................................................................ 3 Chapter 2 literature review .......................................................................................................... 5 The potential role of bacterial proteases in caries and periodontitis pathogenesis ................ 5 2.1 ABSTRACT.............................................................................................................................. 5 2.2 INTRODUCTION ..................................................................................................................... 6 2.3 TOOTH AND SUPPORTING STRUCTURES ................................................................................ 8 2.4 BACTERIAL ASSOCIATED ORAL DISEASES .......................................................................... 15 2.5 BACTERIAL PROTEASES ...................................................................................................... 21 2.6 CONCLUSIVE REMARKS ....................................................................................................... 31 Chapter 3 manuscript ............................................................................................................... 33 Streptococcus mutans proteolytic activity degrade dentinal collagen..................................... 33 3.1 ABSTRACT............................................................................................................................ 33 3.2 INTRODUCTION ................................................................................................................... 35 3.3 MATERIALS AND METHODS ................................................................................................. 36 3.3.1 generic and specific mmp-like activity of s. Mutans ua159 ......................................... 36 3.3.2 soluble type i collagen degradation by s. Mutans ua159............................................. 37 3.3.3 dentinal collagen degradation by s. Mutans ua159 and its discrete fractions ............ 38 3.4 RESULTS .............................................................................................................................. 39 3.4.1 the generic and specific mmp-like activity of s. Mutans ua159 ................................... 39 3.4.2 soluble type i collagen degradation by s. Mutans ua159............................................. 40 3.4.3 dentinal collagen degradation by s. Mutans ua159 and its discrete fractions ............ 41 3.4 DISCUSSION ......................................................................................................................... 42 3.5 CONCLUSION ....................................................................................................................... 47 Chapter 4 manuscript ............................................................................................................... 48 Characterization of proteolytic activity and identification of responsible proteolytic enzymes of streptococcus mutans towards dentinal collagen................................................... 48 4.1 ABSTRACT............................................................................................................................ 48 4.2 INTRODUCTION ................................................................................................................... 50 4.3 MATERIALS AND METHODS ................................................................................................. 51 4.3.1 characterization of proteolytic activity of intracellular proteins of s. Mutans ............ 51 4.3.2 verification of dentinal collagen degradation by intracellular proteins of s. Mutans using sds-page and mass spectrometry ................................................................................. 52 iv 4.3.3 bioinformative analysis of putative genes of collagen-degrading proteases in s. Mutans ua159........................................................................................................................ 53 4.3.4 protein identification of putative collagen-degrading proteases in s. Mutans ua 159 54 4.3.5 cloning, expression and purification of bacterial collagen-degrading proteases ....... 54 4.3.6 degradation of dentinal collagen by smu_759, smu_761 and smu_1438c .................. 56 4.4 RESULTS .............................................................................................................................. 57 4.4.1 characterization of proteolytic activity of intracellular proteins of s. Mutans ............ 57 4.4.2 verification of dentinal collagen degradation by intracellular proteins of s. Mutans using sds-page and mass spectrometry ................................................................................. 58 4.4.3 bioinformative analysis of putative genes of collagenolytic/gelatinolytic proteases in s. Mutans ua159........................................................................................................................ 61 4.4.4 protein identification of putative collagen-degrading proteases in s. Mutans ua 159 61 4.4.5 cloning, expression and purification of bacterial collagenolytic/gelatinolytic proteases ............................................................................................................................................... 61 4.4.6 degradation of dentinal collagen by smu_759, smu_761 and smu_1438c .................. 62 4.5 DISCUSSION ........................................................................................................................