INFLUENCE OF PATHOGENIC BACTERIAL DETERMINANTS ON GENOME STABILITY OF EXPOSED INTESTINAL CELLS AND OF DISTAL LIVER AND SPLEEN CELLS Paul S. Walz B.Sc. Hons., University of Western Ontario, 2009 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfillment of the Requirements for the Degree MASTERS OF SCIENCE Department of Biology University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Paul S. Walz, 2011 INFLUENCE OF PATHOGENIC BACTERIAL DETERMINANTS ON GENOME STABILITY OF EXPOSED INTESTINAL CELLS AND OF DISTAL LIVER AND SPLEEN CELLS Paul S. Walz Approved Dr. Igor Kovalchuk, Co-Supervisor, Department of Biological Science, MD, PhD Date Dr. Olga Kovalchuk, Co-Supervisor, Department of Biological Science, MD, PhD Date Dr. Brent Selinger, Thesis Committee Member, Department of Biological Science, PhD Date Dr. James Thomas, Thesis Committee Member, Department of Biological Science, PhD Date Dr. Lesley Brown, Thesis Committee Member, Department of Kinesiology & Phys. Ed., PhD Date Dr. Elizabeth Schultz/Dr. Theresa Burg, Chair, Department of Biological Science, PhD Date ABSTRACT Most bacterial infections can be correlated to contamination of consumables such as food and water. Upon contamination, boil water advisories have been ordered to ensure water is safe to consume, despite the evidence that heat-killed bacteria can induce genomic instability of exposed (intestine) and distal cells (liver and spleen). We hypothesize that exposure to components of heat-killed Escherichia coli O157:H7 will induce genomic instability within animal cells directly and indirectly exposed to these determinants. Mice were exposed to various components of dead bacteria such as DNA, RNA, protein or LPS as well as to whole heat-killed bacteria via drinking water. Here, we report that exposure to whole heat-killed bacteria and LPS resulted in significant alterations in the steady state RNA levels and in the levels of proteins involved in proliferation, DNA repair and DNA methylation. Exposure to whole heat-killed bacteria and their LPS components also leads to increased levels of DNA damage. iii ACKNOWLEDGMENTS I wish to thank my supervisors, Dr. Igor Kovalchuk and Dr. Olga Kovalchuk for their guidance in helping me complete my research for my thesis. I also wish to extend my gratitude to my committee members; Dr. Brent Selinger, Dr. James Thomas and Dr. Leslie Brown for their helpful advice and direction. I extend my appreciation to my fellow lab members and staff for their assistance and friendship, as well as the Administration of the University of Lethbridge and the Graduate Students Association for their support. To Dr. Daniel Weeks, I thank you for your continued guidance and support. Finally, to my parents and Jacqueline Ziehl, for their never ending love and support during this time. “Wisdom is not a product of schooling but of the life-long attempt to acquire it”. – Albert Einstein iv Table of Contents ABSTRACT ....................................................................................................................... iii ACKNOWLEDGMENTS ................................................................................................. iv Table of Contents ................................................................................................................ v List of Tables ...................................................................................................................... x List of Abbreviations ........................................................................................................ xii CHAPTER 1: GENERAL INTRODUCTION ................................................................... 1 1.1. Escherichia coli O157:H7 ..................................................................................... 1 1.2 Sources of Bacterial Contamination ...................................................................... 3 1.2.1. Sources of Exposure .......................................................................................... 3 1.2.2. Sources of Contamination ................................................................................. 6 1.2.3. Preventative Methods ........................................................................................ 7 1.2.4. Thermal Treatment ............................................................................................ 8 1.4. Carcinogenesis Induced by Bacterial Infection ...................................................... 10 1.4.1. History ............................................................................................................. 10 1.4.2. Viral Infections and Carcinogenesis ................................................................ 13 1.4.3. Bacterial Infection and Carcinogenesis ........................................................... 14 1.4.4. Promotion of Proliferation .............................................................................. 18 1.4.5. Suppression of Apoptosis ................................................................................ 20 1.4.6. Evasion of the Immune System ....................................................................... 21 1.4.7. Chronic Inflammation...................................................................................... 24 1.5 Epigenetic Alteration and Mechanisms of Genome Instability .............................. 25 1.5.1. γH2AX ............................................................................................................. 26 1.5.2. DNMT1 ........................................................................................................... 27 1.5.3. DNMT3A and DNMT3B ................................................................................ 29 1.5.4. MeCP2 ............................................................................................................. 30 1.6 Possible Role of Bacterial Infection in Inducing Genomic Instability.................... 31 1.7 Hypothesis ............................................................................................................... 35 v Chapter 2: Genomic instability in liver cells caused by an LPS-induced bystander-like effect ................................................................................................................................. 40 Abstract ......................................................................................................................... 40 2.1. Introduction ............................................................................................................ 41 The purpose of this study was to analyze of genome stability of an indirect target organ (liver) in mice following digestive tract exposure to DNA, RNA, protein or LPS extracted from heat-killed bacteria. Another objective was to analyse the ability of the mouse organs to react to initial exposure to bacteria/bacterial components and return to a physiological level comparable to the control. ........................................................... 42 2.2. Methods .................................................................................................................. 43 2.2.1. Animal Model .................................................................................................. 43 2.2.2. DNA extraction................................................................................................ 44 2.2.3. RNA Extraction ............................................................................................... 44 2.2.4. Bacterial Protein Extraction ............................................................................. 44 2.2.5. Lipopolysaccharide Extraction ........................................................................ 45 2.2.6. mRNA expression analysis and semi-quantitative RT-PCR ........................... 46 2.2.7. Immunohistochemical Analysis ...................................................................... 47 2.2.8. Western Blot Analysis ..................................................................................... 49 2.2.9. Statistical analysis............................................................................................ 51 2.2.10. Image J ........................................................................................................... 51 2.3. Results .................................................................................................................... 52 2.3.1. Experimental set-up and tissue selection for experiment ................................ 52 2.3.2. Exposure to LPS from heat-killed bacteria leads to increased expression of PCNA in liver cells .................................................................................................... 52 2.3.3. Exposure to LPS from heat-killed bacteria leads to increased levels of γH2AX ................................................................................................................................... 53 2.3.4. Exposure to LPS from heat-killed bacteria leads to an increase in the frequency of DNA damage in the liver tissue ........................................................... 54 2.3.5. Exposure to LPS from heat-killed bacteria leads to an increase in expression of maintenance and de novo DNA methylation enzymes .............................................. 55 2.3.6. Exposure to LPS or whole heat-killed bacteria leads to alterations in mRNA expression within liver tissues ..................................................................................