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University of Florida Thesis Or Dissertation Formatting STREPTOCOCCUS MUTANS OMZ175 INVASION OF HUMAN TISSUES IN CULTURE By EDITH MARION SAMPSON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014 © 2014 Edith Marion Sampson To my family ACKNOWLEDGMENTS I thank my husband, children, and extended family for their support. I thank my colleague and partner in science, Dr. Priscilla Phillips, for daring me to chase after my dreams. I thank Drs. Paulette Robinson, Leticia Reyes, and Brian Bainbridge for their support and kindness. I thank the APF laboratory past and present lab members, including Jacob Burks, Joan Whitlock, Amandeep Chadda, Dr. Amanda Barrett, and Ryan Chastain-Gross for their support and friendship. I thank my undergraduate research assistants, especially Ionut “Johnny” Albu, Bryce Bergeron, Whitney Swonger, Meaghan Foti, and Maria Zimmerman for their time and attention to detail. I thank my mentor, Dr. Ann Progulske-Fox, for her example of a successful academic scientist and for guiding me through thoughtful conversation and constructive criticism. I thank my committee for their high expectations and guidance: Dr. Bill Dunn for his criticism and guidance during lab meetings on my research; Dr. Bob Burne for sharing his knowledge on streptococci and asking hard questions; Dr. Alex Lucas for sharing her knowledge of the cardiovascular system and cardiovascular research; and Dr. Scott Grieshaber for his assistance with confocal microscopy and criticism of the literature and my data. I thank our collaborators Dr. Henry Baker and Cecilia Lopez for completing the microarray chemistry and data analysis, and I thank Dr. Jacqueline Abranches, James Miller, and Dr. Jeanne Brady for sharing bacterial strains, specimens, reagents, and ideas. I would like to thank Karen Kelley, Kim Backer-Kelley, and Neal Benson at the ICBR core facilities. Finally, I would like to thank the Oral Biology T32DE-007200 Training Grant and the Basic Microbiology and Infectious Diseases (BMID) T32AI-7110- 29 Training Grant for the financial support to complete this work. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 13 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 15 Introduction ............................................................................................................. 15 Streptococcus mutans ............................................................................................ 17 S. mutans and the Oral Cavity ................................................................................ 23 S. mutans and Cardiovascular Disease .................................................................. 27 Circulatory System ........................................................................................... 36 Arteries ............................................................................................................. 36 Endothelial Cells ............................................................................................... 37 Pathogen-Host Interaction ...................................................................................... 40 Summary ................................................................................................................ 42 2 STREPTOCOCCUS MUTANS OMZ175 INVASION OF HUMAN ORAL AND CARDIOVASCULAR CELLS .................................................................................. 49 Introduction ............................................................................................................. 49 Materials and Methods............................................................................................ 52 Cell Culture ....................................................................................................... 52 Bacterial Culture ............................................................................................... 53 Attachment Assay ............................................................................................ 53 Antibiotic Protection Assays ............................................................................. 54 Transmission Electron Microscopy (TEM) ........................................................ 55 Relative Real-Time PCR .................................................................................. 56 Host Cell Cycling .............................................................................................. 58 Bacterial Intercellular Spreading ....................................................................... 59 Gene Expression Analysis ................................................................................ 59 Statistical Analysis ............................................................................................ 61 Results .................................................................................................................... 61 S. mutans OMZ175 Invades Human Oral Cells in Culture. .............................. 61 S. mutans Does Not Replicate Intracellularly. .................................................. 63 Intracellular S. mutans Exit the Host and Remain Infectious. ........................... 65 5 Cnm Affects the Expression of Endothelial Cell Cytoskeleton Linked Proteins. ........................................................................................................ 66 S. mutans OMZ175 Invades Human Coronary Artery Smooth Muscle Cells. ... 67 Discussion .............................................................................................................. 68 3 GENE EXPRESSION CHANGES IN HCAEC DURING INFECTION WITH STREPTOCOCCUS MUTANS OMZ175 ................................................................ 92 Introduction ............................................................................................................. 92 Materials and Methods............................................................................................ 95 Preparation of Confluent Human Cells in Culture. ............................................ 95 Bacterial Culture ............................................................................................... 95 Gene Expression Analysis ................................................................................ 95 RT2 Profiler™ PCR Array ................................................................................. 97 RT-PCR Validation ........................................................................................... 97 Enzyme Linked Immunosorbant Assay (ELISA) ............................................... 98 Caspase Activity Assay .................................................................................... 99 Assays to Determine the Ratio of Necrosis/Apoptosis ..................................... 99 Microscopy Analysis of Apoptosis .................................................................. 100 FACS Analysis of Apoptosis ........................................................................... 101 Results .................................................................................................................. 101 Microarray Summary ...................................................................................... 101 Ontology Analysis ........................................................................................... 102 RT-PCR Validation of Microarray ................................................................... 103 Cytokines ........................................................................................................ 103 Cytoskeletal Elements and Adhesion Molecules ............................................ 105 Apoptosis ........................................................................................................ 107 Discussion ............................................................................................................ 110 4 DISCUSSION AND FUTURE DIRECTIONS ........................................................ 140 General Summary and Discussion ....................................................................... 140 Route from Oral Cavity to Cardiovascular Tissues ......................................... 140 Mechanisms of Dissemination ........................................................................ 142 Endothelial Response to S. mutans Infection ................................................. 142 Future Work .......................................................................................................... 144 Introduction ..................................................................................................... 144 Entry and Intracellular Trafficking ................................................................... 145 Role of Cnm in S. mutans Entry ....................................................................
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