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The Role of Collagen Interacting Proteins in the Pathogenesis of Filifactor Alocis Ozioma Salomey Chioma

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The Role of Collagen Interacting Proteins in the Pathogenesis of Filifactor Alocis Ozioma Salomey Chioma Loma Linda University TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works Loma Linda University Electronic Theses, Dissertations & Projects 6-2016 The Role of Collagen Interacting Proteins in the Pathogenesis of Filifactor alocis Ozioma Salomey Chioma Follow this and additional works at: http://scholarsrepository.llu.edu/etd Part of the Medical Cell Biology Commons, Medical Genetics Commons, and the Medical Microbiology Commons Recommended Citation Chioma, Ozioma Salomey, "The Role of Collagen Interacting Proteins in the Pathogenesis of Filifactor alocis" (2016). Loma Linda University Electronic Theses, Dissertations & Projects. 368. http://scholarsrepository.llu.edu/etd/368 This Dissertation is brought to you for free and open access by TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. It has been accepted for inclusion in Loma Linda University Electronic Theses, Dissertations & Projects by an authorized administrator of TheScholarsRepository@LLU: Digital Archive of Research, Scholarship & Creative Works. For more information, please contact [email protected]. LOMA LINDA UNIVERSITY School of Medicine in conjunction with the Faculty of Graduate Studies ____________________________ The Role of Collagen Interacting Proteins in the Pathogenesis of Filifactor alocis by Ozioma Salomey Chioma ____________________________ A Dissertation Submitted in Partial Satisfaction of the Requirements for the Degree Doctor of Philosophy in Microbiology and Molecular Genetics ____________________________ June 2016 ©2016 Ozioma Salomey Chioma All Rights Reserved Each person whose signature appears below certifies that this dissertation in his opinion is adequate in scope and quality, as a dissertation for the degree of Doctor of Philosophy. , Chairman Hansel M. Fletcher, Professor of Microbiology Mark S. Johnson, Associate Research Professor of Microbiology Yiming Li, Professor of Restorative Dentistry Christopher Perry, Assistant Professor of Biochemistry Ubaldo Soto, Assistant Research Professor of Microbiology Aruni Wilson, Associate Research Professor of Microbiology iii ACKNOWLEDGEMENTS I thank God for providing the unique opportunity to follow my dreams of becoming a medical researcher, and for helping me contribute meaningfully to the field of oral microbiology. I thank my mentor, Dr. Hansel Fletcher (Professor & Vice Chair Division of Microbiology of Microbiology), for being an awesome mentor, and encouraging me throughout my graduate school journey. I especially thank him for sharing his expertise in the field of oral microbiology with me. Thank you to all my committee members for providing intellectual guidance on this project. I thank my parents and sisters, for all their prayers and support, and Adé for being my best friend all these years. I thank all my lab colleagues, for all the humor, drama, brain storming and for sharing their skills and knowledge. Thank you Darysbel Garcia, for being my PhD study- buddy and confidante. It wasn’t easy, but we made it, and you are truly amazing. iv CONTENT Approval Page .................................................................................................................... iii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii List of Filifactor alocis Strains and Mutants in This Study............................................. xvi Abstract of the Dissertation ............................................................................................ xvii Chapter 1. Introduction ..............................................................................................................1 Periodontal Disease ............................................................................................1 Risk Factors of Periodontal disease ...................................................................7 Gender and Age ...........................................................................................8 Genetics and Hormones ...............................................................................8 Medications ..................................................................................................9 Lifestyle Choices .........................................................................................9 Diabetes......................................................................................................11 Diagnosis and Treatment of Periodontal Disease ............................................12 Systemic Diseases Associated with Periodontal Disease ................................13 Cardiovascular Disease ..............................................................................13 Pre-term Low Birth Weight Babies ...........................................................14 Osteoporosis ...............................................................................................15 Respiratory Disease ...................................................................................16 Filifactor alocis ................................................................................................16 Role of F. alocis in Periodontal Disease ....................................................17 Virulence Attributes of F. alocis ...............................................................22 Proteases ..............................................................................................22 Biofilm Formation ...............................................................................25 v Oxidative Stress Resistance .................................................................26 Macrophage and Neutrophil Evasion...................................................27 Amino Acid Metabolism......................................................................27 Adhesion and Invasion Related Proteins .............................................28 Collagen ...........................................................................................................31 Role of Bacterial Collagen-Related Proteins in Pathogenesis ...................32 Collagen-Binding/ Collagen-Adhesin Proteins..........................................33 Peptidase U32 Family of Bacterial Collagenases ......................................35 Collagen Related Proteins of F. alocis ......................................................38 F. alocis: Host-Pathogen Interaction ...............................................................39 Apoptosis ...................................................................................................41 Extrinsic Apoptotic Signaling Pathway ...............................................45 Intrinsic Apoptotic Signaling Pathway ................................................47 Perforin/ Granzyme Apoptotic Signaling Pathway..............................47 Anoikis .................................................................................................48 Aims .................................................................................................................51 References ........................................................................................................52 2. Filifactor alocis Collagenase Can Modulate Apoptosis Of Normal Oral Keratinocytes .........................................................................................................88 Summary ..........................................................................................................89 Introduction ......................................................................................................90 Materials and Methods .....................................................................................92 Bioinformatics analysis ..............................................................................92 Bacterial strain culture conditions .............................................................92 Epithelial cell culture conditions ...............................................................93 Purification of PrtFAC in E. coli ...............................................................93 SDS-PAGE and immunoblotting ...............................................................95 Binding of PrtFAC protein to collagen ......................................................95 Collagenase assay ......................................................................................96 Induction of cell death ...............................................................................96 Caspase 3 and 7 activity assay ...................................................................97 RNA isolation and real-time quantitative PCR analysis ............................97 Flow cytometric apoptosis analysis ...........................................................98 Statistical analysis ......................................................................................99 Results………………………………………………………………………103
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