Novel Functions of Anthrax Lethal Toxin A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at George Mason University By Yian Kim Tan Bachelor of Science University of Queensland, 1998 Director: Charles Bailey, Distinguished Professor Department of Molecular and Microbiology Spring Semester 2009 George Mason University Fairfax, VA Copyright 2009 Yian Kim Tan All Rights Reserved ii DEDICATION This is dedicated to my loving wife Karen and my wonderful son Gabriel. iii ACKNOWLEDGEMENTS I would like to express my deep gratitude to the following individuals whose unwavering support and faith were critical to the completion of this research. First, I would like to thank Dr. Charles Bailey for his valuable insights and intellectual support. Dr. Aigou Wu who provided the scientific initiatives that formed the basis of this study and is always so generous with his guidance, feedback and support. Dr. Serguei Popov who provided invaluable scientific comments and suggestions on my research. It is a honour to have them as members of my committee. A special thank you to Dr. Ken Alibek for his mentorship and for allowing me the opportunity to tap into his expertise. I wish to acknowledge the past and present employees at AFG Biosolutions, Inc. namely, Svetlana, Darya, Hao, Caroline and Lena. As a result of their assistance, my research was able to progress smoothly. Finally, I want to thank my family for standing by me every step of the way. To my parents who cheered me on despite the long periods of separation. To my wife, Karen, for being my best friend and critic. To my son, Gabriel, who brought immense joy and laughter to my life. iv TABLE OF CONTENTS Page List of Tables………………………………………………………………………..…vii List of Figures…………………………………………………………………………viii List of Abbreviations…………………………………………………………………… x Abstract................................................................................................................………xii 1. Introduction and Literature Review........................................................................…1 1.1. Introductory Overview .......................................................................................1 1.1.1. Discovery of Anthrax..................................................................................1 1.1.2. Epidemiology .............................................................................................2 1.1.3. Anthrax as a Bioweapon .............................................................................4 1.2. Bacteria Pathogenesis .........................................................................................7 1.2.1. Microbiology..............................................................................................7 1.2.2. Virulence Factors of Bacillus anthracis .....................................................8 1.2.3. Translocation of Lethal Toxin and Edema Toxin into cytoplasm ..........12 1.3. Clinical Manifestation and Diagnosis...............................................................15 1.4. Prophylaxis and Treatment ................................................................................20 1.5. Study Objectives ................................................................................................21 2. Induction of Autophagy by Lethal Toxin .................................................................23 2.1. Introduction ......................................................................................................23 2.2. Materials and Methods......................................................................................40 2.2.1. Cells and Reagents...................................................................................40 2.2.2. Bacterial Culture Supernatant Preparation...............................................40 2.2.3. PCR Detection of Anthrax Chromosome and pXO2...............................41 2.2.4. Stable Transfection of RAW 264.7 Cells ................................................41 2.2.5. Immunoblotting........................................................................................44 2.2.6. Fluorescence Microscopy and Punctuate Count......................................44 2.2.7. Acidic Vacuoles Staining.........................................................................45 2.2.8. Cell Viability.............................................................................................45 2.2.9. Statistical Analysis...................................................................................46 2.3. Results...............................................................................................................46 2.3.1 Acidic Orange Staining Showed Increased Acidic Vacuoles....................46 2.3.2. Increased GFP-LC3 Punctuate When Cells Were Treated with LT.........48 2.3.3. Conversion of LC3-I to LC3-II.................................................................53 2.3.4. Sterne and Delta Ames Strain Contain other Autophagy inducing compound............................................................................................................55 v 2.2.5. Autophagy Inhibitor May Increase Cell Death.........................................56 2.3.3. Discussion.......................................................................................................59 3. Lethal Toxin and Reactive Oxidative Species Production .......................................64 3.1. Introduction Overview......................................................................................64 3.2. Materials and Methods......................................................................................67 3.2.1. Spores Preparation ....................................................................................67 3.2.2. Cell Culture...............................................................................................67 3.2.3. Blood Isolation..........................................................................................67 3.2.4. ROS Production ........................................................................................68 3.2.5. Apoptosis Assay........................................................................................69 3.2.6. Murine Anthrax Model .............................................................................70 3.2.7. Statistical Analysis....................................................................................71 3.3. Results................................................................................................................72 3.3.1. Induction of ROS by LT ...........................................................................72 3.3.2. STIMAL Protection of PBMCs against Apoptosis...................................79 3.3.3. Murine Anthrax Model .............................................................................82 3.4. Discussion..........................................................................................................86 4. Neutrophils Chemotaxis............................................................................................91 4.1. Introduction........................................................................................................91 4.2. Materials and Methods.......................................................................................93 3.3.3. Human Cells Preparation and Activation .................................................93 3.3.3. RNA Isolation ...........................................................................................93 3.3.3. RNase Protection Assay............................................................................94 3.3.3. Measurement of Chemokines Secretion ...................................................95 3.3.3. Neutrophils Chemotactic Assay................................................................95 4.3. Results................................................................................................................96 3.3.3. Inhibition of LPS and CW-Induced Chemokine by LT............................96 3.3.3. LT Inhibits CW and LT-Induced Transcription of Chemokines .............97 3.3.3. LT-Treated PBMC Does Not Induce Neutrophils Chemotaxis................97 4.4. Discussion........................................................................................................103 5. Concluding Remarks...............................................................................................107 List of References .........................................................................................................109 vi LIST OF TABLES Table Page 1.1. Cellular targets and effects of anthrax toxins ..................................................……14 2.1. Description of autophagy-related genes...................................................................27 2.2. Induction of autophagy by bacteria, viruses and toxins...........................................36 3.1. Bacillus anthracis sterne spores (34F2) were administered to DBA2 mice intraperitoneally .............................................................................................................83 3.2. Protective effect of NAC liposome in combination with ciprofloxacin in DBA2 mice infected with B. anthracis sterne spores. .............................................................. 84 vii LIST OF FIGURES Figure Page 1.1. Geographical distribution of anthrax .................................................................……3 1.2.