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From Escherichia Coli O157:H7 and the Bcl-2 Family of Proteins During Host Cell Programmed Cell Death University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Spring 2013 Interactions of shiga-like toxin-2 (Stx-2) from Escherichia coli O157:H7 and the Bcl-2 family of proteins during host cell programmed cell death Lia K. Jeffrey Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Jeffrey, Lia K., "Interactions of shiga-like toxin-2 (Stx-2) from Escherichia coli O157:H7 and the Bcl-2 family of proteins during host cell programmed cell death" (2013). Doctoral Dissertations. 728. https://scholars.unh.edu/dissertation/728 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. INTERACTIONS OF SHIGA-LIKE TOXIN-2 (STX-2) FROM ESCHERICHIA COLI 0157:H7 AND THE BCL-2 FAMILY OF PROTEINS DURING HOST CELL PROGRAMMED CELL DEATH BY LIA K. JEFFREY B.S. University of Massachusetts at Amherst, 1999 DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment the Requirements for the Degree of Doctor of Philosophy in Microbiology May, 2013 UMI Number: 3572946 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Di!ss0?t&iori Piiblist’Mlg UMI 3572946 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ALL RIGHTS RESERVED ©2013 Lia K. Jeffrey This dissertation has been examined and approved. Dissertation Director, Dr. Frank o. Rodgers Professor Department of Molecular, Cellular, and Biomedical Sciences A ay^crvx Dr. Aaron B. Margolir Professor Department of Molecular, Cellular, and Biomedical Sciences Dr. Paul C. Tsang Q Professor Department of Molecular, Cellular, and Biomedical Sciences Dr. Cheryl A. Whistler 4 r Associate Professor Department of Molecular, Cellular, and Biomedical Sciences %0 Dr. Andrew Laudano Associate Professor Department of Molecular, Cellular, and Biomedical Sciences Date DEDICATION I would like to dedicate this dissertation to all of my friends and family who supported me through this process, and to my late father, Edward J. Nalewak Jr., who inspired me to be the first in my family to work towards a doctoral degree. I would especially like to thank my husband, John who was my grounding factor and kept me focused on the task at hand, my mom, Denise Tallarida who always would be there to listen when my experiments didn’t go as planned, and Dr. Rodgers for giving me a truly wonderful education and mentorship I will never forget. ACKNOWLEDGEMENTS I would like to acknowledge Dr. Rodgers for the opportunity to earn this degree and for all of his time and effort he spent mentoring me. I would also like to acknowledge the members of my committee for their help and guidance throughout my research project, and Adele Marone for being my designated phlebotomist. In addition, I would like to thank my colleagues at EMD Millipore, especially Kerry Roche-Lentine for supporting and allowing me the flexibility to perform my research on-site during the latter stages of these studies while maintaining my full-time position. To all the undergraduates who worked with me on projects related to the research presented in this thesis, thank you. Your help was very much appreciated during the many hours spent performing western blots, ELISA assays, and other experimentation. I would also like to thank Kate Stefani for her friendship and support during my graduate studies; you will always be my research lab partner in crime. I would also like to acknowledge the USD A Hatch Grant for supporting and funding this research, the University of New Hampshire’s College of Life Sciences for providing fellowships and scholarships throughout my graduate degree, and the UNH Institutional Biosafety Committee and Institutional Review Board for the Protection of Human Subjects in Research for authorization to perform the recombinant DNA and human aspects of this research. v TABLE OF CONTENTS DEDICATION......................................................................................................... iv ACKNOWLEDGEMENTS...................................................................................... v LIST OF CHAPTERS.............................................................................................. vi LIST OF TABLES.................................................................................................... xiii LIST OF FIGURES.................................................................................................. xiv ABSTRACT............................................................................................................. xviii CHAPTER PAGE 1. INTRODUCTION TO ESCHERICHIA COLI0157:H7 INFECTION AND HOST CELL RESPONSES 1.0 Microorganism Description ....................................................................... 1 2.0 Overview of Infection ................................................................................ 6 3.0 Epidemiology ............................................................................................. 7 4.0 Virulence Factors ....................................................................................... 11 4.1 Shiga-like Toxins ....................................................................................... 11 4.2 Adhesins..................................................................................................... 14 4.3 Lipopolysaccharide .................................................................................... 15 4.4 Enterohemolysin ........................................................................................ 15 4.5 Extracellular Serine Protease ..................................................................... 16 4.6 Enterotoxin ................................................................................................ 17 5.0 Pathogenesis of E. coli 0157:H7 Infection .............................................. 17 6.0 Host Response to E. coli 0157:H7 Infection ........................................... 23 7.0 Mechanisms of Eukaryotic Cell Death ...................................................... 27 INTERACTIONS BETWEEN THE SHIGA-LIKE TOXINS AND BCL-2 PROTEIN FAMILY 1.0 Abstract...................................................................................................... 36 2.0 Introduction ............................................................................................... 37 3.0 Hypothesis................................................................................................. 39 4.0 Materials and Methods .............................................................................. 39 4.1 Stx-2 Protein Sequence Analysis ............................................................... 39 4.2 Purification of Stx-2 from Escherichia coli 0157:H7 strain 90-2380 ...... 40 4.3 Stx-2 Characterization............................................................................... 41 4.3.1 Native Analysis of Semi-Purified Stx-2 .................................................... 41 4.3.2 SDS-PAGE Analysis of Semi-Purified Stx-2 ............................................ 42 4.3.3 Western Blot Analysis of Semi-Purified Stx-2 .......................................... 44 4.3.4 Cleavage Analysis of Semi-Purified Stx-2 and A1 Fragment Generation. 45 4.3.5 Biological Activity Verification of Semi-Purified Stx-2 ............................ 46 4.3.6 Quantitation of Semi-Purified Stx-2 .......................................................... 47 4.4 Interactions of Stx-2 with Caco-2 Cells ..................................................... 47 4.4.1 Susceptibility of Caco-2 Cells to Stx-2 ...................................................... 48 4.4.2 Protein Analysis of Caco-2 Cells ................................................................ 48 4.4.3 Interactions of Bel-2 (from Caco-2 Cells) and Stx-2 ................................. 51 4.4.4 Caco-2 Cell Lysate Preparation for Co-Immunoprecipitation.................. 52 4.4.5 Co-Immunoprecipitation of Stx-2 and Bcl-2 Family Proteins .................. 53 5.0 Results...................................................................................................... 56 5.1 Stx-2 Protein Sequence Analysis ............................................................... 56 5.2 Stx-2 Characterization.............................................................................. 58 5.2.1 Native Analysis of Semi-Purified Stx-2 .................................................... 58 5.2.2 SDS-PAGE and Western Blot Analysis of Semi-Purified Stx-2 .............. 58 5.2.3 Cleavage Analysis of Semi-Purified Stx-2 and A1 Fragment Generation. 63 5.2.4 Biological Activity Verification of Semi-Purified Stx-2 ........................... 66 5.2.5 Quantitation of Semi-Purified Stx-2 ........................................................
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